Long-term Mg2+ imaging during apoptosis using a HaloTag-coupled Mg2+ probe demonstrated a Mg2+ concentration increase caused by dissociation of Mg2+ from ATP. 2+ plays important roles in many physiological processes. However, the underlying molecular mechanisms, especially in the apoptotic pathway, remain unclear due to the diffusion of Mg2+ probes, which hinders long-term imaging in specific organelles. We developed an immobilized Mg2+ probe, MGH, which is covalently conjugated with the HaloTag protein in various organelles. HaloTag-coupled MGH enabled long-term imaging of intracellular local Mg2+ dynamics for 24 h. To exploit this remarkable property, MGH was applied to the investigation of intracellular Mg2+ dynamics during apoptosis. Time-lapse imaging revealed an increase in the Mg2+ concentration after apoptotic cell shrinkage. Combined imaging analyses of intracellular Mg2+ and ATP concentrations strongly suggested that this Mg2+ concentration increase was caused by the dissociation of Mg2+ from ATP, along with a decrease in the intracellular ATP concentration. Thus, this protein-coupled Mg2+ probe could be a new chemical tool to elucidate intracellular Mg2+ dynamics with high spatiotemporal resolution.Mg However, the intracellular free Mg2+ concentration ([Mg2+]i) is maintained between 0.5 and 1 mM, because most Mg2+ forms complexes with nucleotides, such as ATP or DNA.2+ plays vital roles in a variety of physiological processes such as regulation of enzyme activities, stabilization of nucleotides, and cell proliferation.2+ homeostasis is tightly regulated by Mg2+ transporters and Mg2+ channels and buffered by intracellular ATP.2+ plays a role as a secondary messenger in T cells.2+ homeostasis is involved in several disorders including diabetes, hypertension, Parkinson’s disease, and cancer.Mg2+ being obviously important for regulating cellular functions, regulation of the underlying molecular mechanisms of [Mg2+]i still remains unclear.2+ dynamics are unclear is apoptosis. Apoptosis is a programmed cell death process occurring over several hours to eliminate damaged cells. While Ca2+ plays very important roles in regulating the apoptotic process,2+]i have also been observed in apoptotic cells, for example hepatocytes treated with glycodeoxycholate2+ dynamics during apoptosis have not been continuously visualized due to the lack of suitable analytical tools. To date, commercially available Mg2+ fluorescent probes, such as magnesium green and Mag-Fura-2, have often been utilized to detect free Mg2+ in cells. However, these probes tend to quickly diffuse over the entire cytoplasm and leak out of the cells via anion transporters.2+ concentration changes over long time periods, resulting in insufficient information regarding the intracellular Mg2+ dynamics in apoptotic cells.Despite Mg2+ imaging, various Mg2+ probes have been developed. Recently, genetically encoded fluorescent protein-based Mg2+ sensors, MagFRET2+ sensors have not yet been developed.To improve the spatial and temporal resolution of Mg2+ probes with improved fluorescence properties and intracellular behaviors have been developed for the exploration of intracellular Mg2+ dynamics. A chemical Mg2+ sensor functionalized with a lipophilic cationic alkylphosphonium group, Mag-mito, showed targetability to mitochondria.2+ concentration in mitochondria by calculating the ratio of fluorescence signals obtained using two different excitation wavelengths. Mag-mito successfully detected an increase in free Mg2+ in mitochondria in the early stages of staurosporine-induced apoptosis. However, this probe diffused from the mitochondria to the cytosol after 40 min of the compound treatment due to the depolarization of the mitochondrial membrane during apoptosis.2+ concentration in various other organelles, novel fluorescent probes with different targeting moieties must be developed.Therefore, small molecule-based Mg2+ probes has rarely been reported. One example is KMG-104-AsH,2+ sensors in the KMG series.2+ concentration changes. However, long-term imaging of more than 4 h has not been carried out, probably owing to the cellular toxicity of an additive (ethanedithiol) that is necessary to reduce nonspecific binding.2+ dynamics in living cells have been pursued.Therefore, a versatile method is to use a protein labeling system, with a pairing of a genetically encoded tag and its specific ligand. So far, several targetable fluorescent sensors have been developed on the basis of protein labeling systems, such as SNAP-tag and HaloTag.2+ probe that can be localized in specific organelles for imaging local Mg2+ dynamics in living cells. Through covalent anchoring of the Mg2+ probe to a tag protein expressed in various cellular compartments, intracellular diffusion and extracellular leakage of the Mg2+ probe were suppressed. This enabled not only the localization of the Mg2+ probe, but also long-term imaging of intracellular Mg2+ dynamics. This approach provided the first long-term imaging of [Mg2+]i during apoptosis.To address these issues, we developed a tag protein-conjugatable Mg2+ dynamics in living cells for a long time, we designed and synthesized a new Mg2+ sensor, MGH, which covalently labels HaloTag , making it able to detect [Mg2+]i changes.2+-unbound state is suppressed by photo-induced electron transfer (PeT),via binding to Mg2+.Here, magnesium green was adopted for Mg2+ chelator part, the fluorophore part, and the HaloTag ligand part. These components were individually synthesized, and were then conjugated to facilitate the synthetic process and optimization of each part. The Mg2+ chelator part (compound 4) was synthesized from o-aminophenol via SN2 reaction with chloroacetic acid, Fischer esterification, nitration, and alkaline hydrolysis (Scheme S1via the Mannich reaction (Scheme S2via the amide condensation reaction of the chelator moiety (compound 6) and chromophore (compound 11), and then it was conjugated with the HaloTag ligand (compound 14) (Scheme S3via Cu(i)-catalyzed azide–alkyne cycloaddition to produce MGH(AM) .MGH is composed of three building blocks for synthesis: the MgScheme S1.36 To imScheme S2.38 Then,Scheme S3 via Cu(iScheme S4. MGH(AM)2+ -based fluorescent probes.2+ imaging, the ability of the probe to discriminate changes in intracellular Ca2+ concentration ([Ca2+]i) is quite important. MGH bound to Ca2+ more strongly (Kd (Ca2+) = 12 μM) than to Mg2+   containing 115 mM KCl and 20 mM NaCl. Although the absorption spectra of MGH did not change significantly with 100 mM Mg2+ , the flue 1.3 mM , which i Fig. S1d. These s+ sensor .2+, HaloTag-MGH has almost the same dynamic range of as that of free MGH, which was derived from the hydrophilic spacer between the Mg2+ sensor part and the HaloTag ligand part. The hydrophilic spacer prevented the interaction between the chromophore and the protein surface of the HaloTag.Kd values of HaloTag-MGH for Mg2+ and Ca2+ were enhanced approximately 2-fold, the affinity of HaloTag-MGH for Mg2+ was still appropriate for visualizing intracellular Mg2+ dynamics.It is likely that spectroscopic properties of fluorescent probes are affected by environmental changes such as binding on the protein surface. Hence, we measured the spectroscopic properties of MGH after incubation with a purified HaloTag protein for 1 h at 37 °C. SDS-PAGE analysis showed almost all MGH covalently bound to HaloTag Fig. S3. Overall11-Halo, or HaloTag. NLS is a nuclear localization signal peptide11 is a Lyn N-terminal sequence (GCIKSKGKDSA), which is used to target proteins to the inner leaflet of cell membrane.Here, we attempted to confirm the subcellular targeting of MGH to a variety of organelles. HEK293T cells were transfected with a plasmid encoding Halo-NLS, Lyn2+ in the ER because the ER contains high concentrations of Ca2+ (hundreds of μM). Therefore, Mg2+-selective probes are necessary for the correct evaluation of changes in Mg2+ concentration in the ER.Subsequently, MGH(AM) was added to HEK293T cells expressing HaloTag in the endoplasmic reticulum (ER) or the mitochondrial matrix. Since these organelles are covered by a lipid bilayer membrane, it was unclear whether fluorescent probes modified with AM esters could penetrate the second lipid bilayer membrane prior to the enzymatic hydrolysis of the AM esters by intracellular esterases. To express HaloTag in ER, the ER signal peptide and retention signal sequences (SEKDEL) were fused with HaloTag . For mitochondrial matrix targeting, HaloTag was fused with the first 36 amino acids of subunit VIII of cytochrome c oxidase (COX8) in tandem to enhance the specificity of mitochondrial localization .2+ imaging in living cells was attempted by using the nucleus-localized MGH. Commercially available small-molecule Mg2+ probes, such as magnesium green, are not suitable for long-term imaging owing to the extracellular leakage via anion transporters.2+ dynamics for more than 1 day.Next, long-term Mg2+, we performed a Mg2+ extrusion experiment using a Mg2+ transporter, ancient conserved domain protein/cyclin M4 (CNNM4). CNNM4 is strongly expressed in the intestinal epithelium and can extrude Mg2+ by stimulating Na+/Mg2+ exchange.2+ buffer. After 1 h, the extracellular solution was exchanged with a Mg2+-free buffer to artificially promote Mg2+ extrusion via CNNM4, and time-lapse fluorescence microscopic images were captured. The fluorescence intensity of nucleus-localized MGH, as well as magnesium green, immediately decreased after Mg2+ depletion from the extracellular buffer  loading to the transfected HEK293 cells. As a result, the fluorescence of MGH quickly decreased after exchanging a buffer containing 40 mM Mg2+ with the Mg2+-free buffer . So far, anti-Fas antibody-mediated [Mg2+]i increase in apoptotic B cells was fragmentally detected for 16 h with a ratiometric Mg2+ probe, Mag-indo-1, by flow cytometric analysis.2+ mobilization along with the apoptotic process. On the other hand, use of MGH simultaneously enables real-time monitoring of [Mg2+]i and changes in cellular morphology over a long time period. However, MGH exhibits an increase in fluorescence intensity without a shift in wavelength. This means that the fluorescence intensity depends not only on Mg2+ concentration but also on other factors such as probe concentration and light intensity. Particularly, probe concentration must be changed during apoptosis, because apoptotic cells show shrinking. Therefore, to exclude the influence of such factors, an internal standard was adopted for calibration. As the internal standard, a red fluorescent HaloTag ligand, Halo-TMR, was used to label HaloTag, and the fluorescence intensity ratio of MGH and Halo-TMR was calculated.Taking advantage of the applicability to long-term imaging, MGH was applied to investigate intracellular Mg2+]i increased from approximately 1 to 1.8 mM during apoptosis  for 30 min and subsequently Halo-TMR for 15 min. Then, the time-lapse imaging experiment was started, and anti-Fas antibody and CHX were added after 30 min. Alexa Fluor™ 350 annexin V conjugate, which binds phosphatidylserine-binding protein, was also added to identify apoptotic cells. As a result, the fluorescence signal ratio of MGH and Halo-TMR increased after the apoptotic cell shrinkage, and then plateaued . Quantits Fig. S6.2+]i during long-term imaging experiments. Actually, long-term microscopic imaging data demonstrated that the fluorescence ratio of MGH/Halo-TMR in HaloTag-expressing HeLa cells without the addition of apoptosis inducers continued to decrease slightly for several hours  = 480 nM).2+ introduced by the ionophore 4-bromo-A23187, in contrast to the increase in the fluorescence intensity of MGH, demonstrating sufficient specificity for Ca2+ .Another concern related to the increase in the fluorescence ratio of MGH/Halo-TMR is equilibration of intracellular and extracellular metal ions due to the loss of cell membrane integrity in the late stages of apoptosis.2+]i increase during apoptosis, we focused on the correlation between [Mg2+]i and intracellular ATP concentration; since most ATP forms a complex with Mg2+ in cells,2+]i is likely to be affected by ATP concentration. In fact, it has been reported that various stimulations, such as cyanide,2+]i with a decrease in intracellular ATP or degradation of ATP to ADP or AMP. These reports suggested a close relationship between Mg2+ and ATP levels. Another previous study showed that intracellular ATP concentration considerably decreased at the late stage of apoptosis.To elucidate the mechanism of [Mg2+]i and ATP concentration in apoptotic cells was desirable, it was not possible because the excitation and emission wavelengths of MGH and ATeam largely overlap. Therefore, the fluorescence ratio changes for [Mg2+]i and intracellular ATP concentration were plotted from 1 h before cell shrinkage  and potassium cyanide (KCN), which are inhibitors of glycolysis and oxidative phosphorylation, respectively.2+]i .2+ transport mechanisms may contribute to the regulation of intracellular Mg2+ homeostasis. Since MGH can be localized on a Mg2+ transporter by tandemly expressed HaloTag, Mg2+ influx via Mg2+ transporters would be visualized using such organelle-localized probes. Further studies using MGH should lead to the elucidation of Mg2+-buffering mechanisms in apoptotic cells.In HeLa cells, the intracellular ATP concentration was estimated at 3–4 mM,s Fig. S6. This re2+ probe, MGH, which covalently binds to HaloTag protein expressed in various cellular compartments. The conjugation of MGH to HaloTag dramatically suppressed the extracellular leakage of MGH, and the Mg2+ sensing ability of MGH was retained for 24 h. These noteworthy properties were successfully applied to long-term visualization of intracellular Mg2+ dynamics during apoptosis. The results demonstrated an increase in Mg2+ concentration after apoptotic cell shrinkage. Subsequent experiments utilizing a FRET-based ATP sensor showed that dissociation of Mg2+ from ATP in apoptotic cells was the source of the increase of Mg2+ concentration. Thus, long-term imaging with a HaloTag-coupled Mg2+ probe provided precise information regarding intracellular Mg2+ dynamics during apoptosis. This study includes the molecular design of a cell-functional probe that involves a synthetic organic molecule and a genetically encoded protein. In addition, the findings in this study should contribute to the understanding of Mg2+-related biology, which includes very wide areas in biology and medicine, and thus the results strongly suggest that this new methodology will be a robust tool for studying Mg2+ dynamics in living systems and diseases.In conclusion, we developed a novel Mg2 were transfected with pcDNA-3.1-(+)-Halo-NLS, pcDNA-3.1-(+)-Lyn11-Halo, pcDNA-3.1-(+)-HaloTag, pKmc-2xCOX8-Halo or pmKate2-Halo-ER plasmids using Lipofectamine 3000, and the cells were incubated at 37 °C for 24 h. Then, the cells were washed three times with HBSS and incubated in FBS-free DMEM containing 3 μM MGH(AM) for 1 h in a CO2 incubator. After washing with HBSS, fluorescence images were captured in DMEM containing 10% FBS using a confocal fluorescence microscope at 37 °C.HEK293T cells maintained in 10% FBS in DMEM at 37 °C in 5% CO2+-loading buffer , including 5 μM MGH(AM) or 2 μM magnesium green, for 1 h at 37 °C. The cells were rinsed once with loading buffer and viewed using an epifluorescence microscope . Fluorescence was measured every 20 s (excitation at 470–490 nm and emission at 505–545 nm) under the control of the MetaMorph software (Molecular Devices). Then, the buffer was changed to a Mg2+-free buffer (MgCl2 in the loading buffer was replaced with 60 mM NaCl). The Mg2+ extrusion experiment 24 h after loading MGH(AM) to HEK293 cells was performed as follows. HEK293 cells were firstly transfected with pcDNA-3.1-(+)-Halo-NLS, and the cells were incubated at 37 °C for 24 h. The cells were incubated with 5 μM MGH(AM) in DMEM (FBS free) for 1 h at 37 °C, and then the cells were transfected with pCMV-CNNM4-FLAG at 37 °C for 24 h. After incubation with Mg2+-loading buffer for 30 min, the cells were rinsed once with loading buffer and viewed using an epifluorescence microscope.HEK293 cells were transfected with pCMV-CNNM4-FLAG and pcDNA-3.1-(+)-Halo-NLS using Lipofectamine 2000, and the cells were incubated at 37 °C for 24 h. Then, the cells were incubated with Mga2 were transfected with the pcDNA-3.1-(+)-HaloTag plasmid using Lipofectamine 3000. After 24 h, the cells were washed twice with HBSS, incubated with 3 μM MGH(AM) for 30 min, and then treated with 50 nM Halo-TMR for 15 min at 37 °C under 5% CO2. After washing with HBSS, fluorescence images were captured in DMEM containing 10% FBS and 5 μL AnnexinV (Alexa Fluor 350) using a confocal fluorescence microscope at 37 °C. Anti-Fas antibody (250 ng mL–1) and cycloheximide (10 μg mL–1) were added 30 min after the imaging started. Change in [Mg2+]i was determined using the following equation:[Mg2+]i = KdQ(R – Rmin)/(Rmax – R),where R is the signal ratio of MGH/Halo-TMR, Rmin is the minimum value of R, Rmax is the maximum value of R, Q is the signal ratio of Halo-TMR under minimum Mg2+ concentration to Halo-TMR under maximum Mg2+ concentration, and Kd is 0.67 mM for Halo-MGH. Rmin and Rmax were calculated after imaging experiments as follows. The apoptotic cells were washed with Mg2+- and Ca2+-free HHBSS buffer, then permeabilized with 20 μg mL–1 digitonin in Mg2+- and Ca2+-free HHBSS for 5 min. After washing the cells with Mg2+- and Ca2+-free HHBSS buffer, Rmin was recorded by incubating the cells in Mg2+- and Ca2+-free HHBSS buffer containing 10 mM EDTA at 37 °C for 10 min. Then, Rmax was recorded by incubating the cells in Ca2+-free HHBSS buffer containing 50 mM MgCl2 at 37 °C for 10 min.HeLa cells maintained in 10% FBS in DMEM at 37 °C in 5% COb2 were transfected with the pcDNA-3.1-(+)-HaloTag and CMV-R-GECO1 plasmids using Lipofectamine 3000. After 24 h, the cells were washed twice with HBSS and incubated with 100 nM Halo-OG for 30 min at 37 °C under 5% CO2. After washing with HBSS, fluorescence images were captured in DMEM containing 10% FBS and 5 μL AnnexinV (Alexa Fluor 350) using a confocal fluorescence microscope at 37 °C.HeLa cells maintained in 10% FBS in DMEM at 37 °C in 5% COc2 were transfected with the pcDNA-3.1-(+)-ATeam plasmid using Lipofectamine 3000. After 24 h, the cells were washed two times with HBSS and then fluorescence images were captured in DMEM containing 10% FBS and 5 μL AnnexinV (Alexa Fluor 680) using a confocal fluorescence microscope at 37 °C.HeLa cells maintained in 10% FBS in DMEM at 37 °C in 5% COHeLa cells were transfected with ATeam or HaloTag plasmids. The cells expressing HaloTag were washed twice with HBSS, incubated with 3 μM MGH(AM) for 30 min, and then treated with 50 nM Halo-TMR for 15 min at 37 °C. After washing with HBSS, fluorescence images were captured in DMEM at 37 °C for 1 h. The 2DG and KCN were added 10 min after the imaging started.There are no conflicts to declare.Supplementary informationClick here for additional data file.


To describe the case of two siblings with severe protein-calorie malnutrition dueto abuse by starvation.The two patients were simultaneously referred to the Hospital Municipal, wherethey were admitted to the Pediatric Gastroenterology clinic of a universityhospital for diagnostic investigation of the cause of severe malnutrition andscreening tests for Celiac Disease, Cystic Fibrosis and Environmental enteropathyamong others. The exams were all normal, and after detailed research on theinteractions of this family, we reached the conclusion that the malnutrition wasdue to abuse by starvation. The children spent approximately two months in thehospital, receiving a high-protein and high-calorie diet, with significantnutritional recovery.Abuse by starvation, although rare, should always be considered of as one of thecauses of child malnutrition and pediatrician should be aware of the child'sdevelopment, as well as the family interactions, to prevent more severenutritional and emotional consequences in the future. Protein-calorie malnutrition in childhood is a worldwide public health problem,especially in countries of low and middle income, being related to more than one thirdof all deaths of infants and children under five years in these countries.The United Nations Children's Fund (UNICEF) recognizes environmental, economic andsocio-political factors as root and underlying causes of malnutrition, with povertyrepresenting the core of the problem.Another less common, but extremely serious cause concerns abuse by starvation, whenparents or caregivers deliberately fail to feed their children, which can lead to riskof death.The aim of this study is to describe the cases of two siblings suffering from severeprotein-energy malnutrition due to abuse by starvation, which characterizes a type ofmistreatment.The two patients were simultaneously referred from the Hospital Municipal de Diadema(HMD), where they were admitted to the Pediatric Gastroenterology Clinic of EscolaPaulista de Medicina for diagnostic investigation of the severe protein-caloriemalnutrition causes.sic); so shecalled the Mobile Emergency Service (SAMU) and there was need for resuscitationmaneuvers (sic). After that, the patient was taken to HMD, where it wasdecided to admit him, together with his older brother, due to a picture of severemalnutrition.The mother reported that her younger son, 4 years and 8 months, was feeling fine athome, when suddenly he went into “respiratory arrest” .After 4 years of age, these measures were again recorded in the vaccination card,which showed evident weight and growth impairment.According to the mother's report, the patient had an adequate diet . She denied the occurrence of diarrhea, constipation, abdominal painand/or distension or any other gastrointestinal symptoms.Regarding the family history, the patient has a brother aged 4 years and 8 monthswith a similar picture. The father is healthy and the mother was followed at thePsychiatry Service, used medications, but had no definitive diagnosis. At the firstvisit, the mother said her name was Maria das Graças; however, in subsequentconsultations, we realized that their children called her Ana Paula. The mother wasinvestigated on suspicion of mistreatment and accompanied the children duringhospitalization at HMD.The family lived in a house with basic sanitation, with running water and sewagesystems. Family income varied from 1 to 5 minimum wages.Z-score=−6.56) and height=87cm (height/ageZ-score=−6.21).On physical examination, the patient showed a regular general status, extremelyemaciated, pale (+/4+), apathetic, with scarce subcutaneous tissue, muscle atrophy ofthe gluteal region and abdominal distension. Weight=8.5kg 1.191U/L; serum glutamic pyruvic transaminase (SGPT) 1.043U/L. These changes wereattributed to a picture of severe protein-calorie malnutrition.At the first consultation, the diagnosis of severe protein-calorie malnutrition wascharacterized and the patient started being investigated for the following causes:celiac disease, cystic fibrosis of the pancreas and environmental enteropathy, amongothers.The following laboratory tests were requested: anti-transglutaminase antibody, sweattest (sodium and chloride in sweat) and upper endoscopy with duodenal biopsy.Laboratory tests were negative for the suspected diagnoses and duodenal biopsydisclosed fingerlike villi and celiac disease was ruled out .The patient was hospitalized for two months at HMD and during the hospitalizationperiod, he received a high-calorie and high-protein diet. The patient showedexcellent nutritional recovery  andconsThe excellent clinical outcome triggered exclusively by a high-calorie andhigh-protein diet reinforced the diagnostic suspicion of mistreatment, withsubsequent progression to severe protein-calorie malnutrition by starvation. Thechild was legally removed from their parents and is, together with his brother, in ashelter.Male patient, aged 4 years and 8 months, born and residing in the municipality ofDiadema, was referred to our service from HMD, where he was hospitalized.The mother reported that the BHU pediatrician started to realize that the patient didnot gain weight after 3 years of age. She denied abdominal distension and diarrhea.She reported that the patient ate adequately and, according to the nutrition teamassessment, it was not possible to identify any protein-calorie restriction inrelation to age.Z-score=−5.73) and height=80cm(height/age Z-score=−6.35).On physical examination, the child showed regular general status, was very emaciated,weight=7.585kg (weight/age At the first consultation, the diagnosis of severe protein-calorie malnutrition wasmade and the patient started to be investigated for the same causes of malabsorptivediseases that his brother was undergoing, with negative test results. The duodenalbiopsy disclosed fingerlike villi and celiac disease was ruled out .The patient remained hospitalized for two months at HMD with his brother, receiving ahigh calorie and high-protein diet, showing excellent nutritional recovery  and 6, wDuring one of the visits to our clinic, when the children were already living in theshelter, we asked the social worker about the fact that her mother sometimes said hername was Maria das Graças and, sometimes, Ana Paula. The social worker informed usthat up until the older brother was aged 2 and a half years, the children and theirparents lived near the rest of the relatives; however, they moved to an unknowndestination and lost contact with the family. In this new home, the patient's mothertold the neighbors her name was Ana Paula.Regarding the children's father, he said that he worked all day and did not realizethat the mother did not feed children adequately. He was considered an accomplice andthe children were kept away from him.,Malnutrition caused by family neglect, which is characterized by a non-deliberatefailure to meet the child's needs, can occur due to ignorance, low socioeconomic statusor even dietary beliefs.On the other hand, abuse by starvation, the cause of malnutrition in our two patients,differs from neglect by the fact that it is characterized as deliberate or maliciousfailure to meet the child's needs.Nancy et al.,Regarding the fathers' role, Krieger,In the national literature, although there are many articles about child abuse, thereare few reports on malnutrition caused by abuse due to starvation. Nudelmann andHalpern,A differential diagnosis that also deserves to be discussed is Munchausen syndrome byproxy, corresponding to a form of child abuse caused by a perpetrator with a psychiatricdisorder that exacerbates, falsifies or promotes clinical histories, laboratoryevidence, can cause physical injuries and induce hospitalizations for unnecessarytherapeutic and diagnostic procedures.In the cases discussed in this article, the mother, who had a psychiatric disorder, toldthe emergency service that her child had a “respiratory arrest”, a fact that was notconfirmed by the team who treated the child.,A noteworthy aspect concerns the morphological findings in the small intestine atoptical microscopy in both patients, considering that the absence of severe injuriescould constitute an apparent paradox. However, the description of morphologicalalterations of the small intestine mucosa in most cases of severe protein-caloriemalnutrition is accompanied by environmental enteropathy.Our patients, in spite of the severe protein-calorie malnutrition by starvation, hadintestinal villi perfectly compatible with normality; the inflammatory infiltrate of thelamina propria was discreet and the villus/crypt ratio was 4/1. Interestingly, ourpatients, as observed in the experimental study of malnourished rats regarding theincreased absorptive function, when given a high-protein and high calorie diet, showedrapid nutritional recovery within a short period of time, which proves that theirdigestive-absorptive functions were kept intact despite the prolonged period of fooddeprivation. These findings demonstrate for the first time in humans, to the best of ourknowledge, that severe protein-calorie malnutrition is not primarily the cause of severedisorders of the digestive-absorptive function: the occurrence of an external factor,such as the environmental contamination, is necessary to trigger all thepathophysiological and symptomatic process described in environmental enteropathy.Regarding the prevalence of bacterial overgrowth associated with environmentalenteropathy in Brazil, some studies have been carried out with the lactulose hydrogenbreath test. A study involving 83 schoolchildren living in the rural, urban areas and aslum in a municipality in the countryside of São Paulo disclosed bacterial overgrowth in7.2% of the assessed children.Protein-calorie malnutrition can be caused by organic diseases, poor social condition,neglect and abuse by starvation. The latter cause is rare, with few reports in theliterature, but it has severe consequences, both nutritional and psychological, for theaffected child. The pediatrician plays a key role in the early detection of these casesand should always be alert to monitor the child's weight/height development, as well asthe family relationships.


Ileal pouch-anal anastomosis surgery can be complicated by anastomotic leaks, leading to the formation of abscess and chronic sinus that have been routinely managed by a surgical approach. We developed the endoscopic needle knife sinusotomy (NKSi) technique, which has become a valid alternative. The basic principle of endoscopic NKSi is dissection and drainage of the sinus through its orifice internally into the lumen of pouch body. The success of NKSi requires an access to the sinus from the pouch side. One of the most challenging situations for NKSi is a closed orifice of the sinus, which leaves an isolated chronic abscess cavity. Here we report a case of complicated presacral sinus with a closed orifice that was not amenable to NKSi, necessitating a CT-guided guide wire placement and subsequent NKSi. Anastomotic leaks after ileal pouch-anal anastomosis (IPAA) surgery for ulcerative colitis (UC) or familial adenomatous polyposis occur in up to 10% of patients . These l3. Subsequently, a diagnostic pouchoscopy was performed and showed normal afferent limb, inlet, tip of J pouch body and cuff. The orifice of the presacral sinus was not identified on endoscopy, even by extensive probing with a soft-tip guide wire. In fact, the pouchoscopy showed a complete normal pouch anastomy and normal mucosa of afferent limb, pouch body, and anal transitional zone. A transanal endoscopic ultrasound was attempted and revealed a thick wall between the pouch body and sinus tract that precluded needle aspiration of the abscess.The patient was a 23-year-old male who presented to our clinic with abdominal pain, hematochezia and coccyx tenderness for five years after IPAA surgery. He was diagnosed as having UC in 2005, which became medically refractory, and he eventually underwent total proctocolectomy and two-stage IPAA in December 2008. Symptoms of abdominal cramping and increased bowel frequency developed immediately after ileostomy closure, which had initially responded to oral metronidazole and tetracycline therapy. At the time of presentation, he had been on long-term ciprofloxacin or metronidazole therapy for ‘pouchitis.’ Pelvic MRI six months prior showed a long sinus tract extending from the pouch anal anastomosis. A repeat MRI confirmed a fluid-containing tract extending from the presacral region anteriorly and encircling the midportion of the pouch above the level of the anastomosis . There wGiven the complexity of this sinus, we opted for CT-guided percutaneous placement of a guide wire to localize the sinus orifice. The sinus tract was localized using a needle from a 15-cm 20-gauge wire localization set and intermittent injection of dilute Omnipaque® 240 mg I/ml  contrast by the experienced radiologist (JV). With the tract identified, a small amount of gas was insufflated into the pouch via rectal catheter, and the needle was advanced into the pouch. The localization wire was advanced through the needle into the pouch, and the needle was removed. The localization wire was then secured to skin. CT imaging confirmed the placement of wire into the distal pouch body .Figure Subsequently, therapeutic pouchoscopy with NKSi was performed, with dissection of the sinus wall along the course of the wire. A 4-cm long sinusotomy was created, with dissected edges being stapled with endoclips to prevent reclosure of the orifice . The guiAnastomotic leaks after IPAA surgery occur in up to 10% of patients, and medical management alone is often futile once an inflammatory tract or abscess forms ,5. EndosThe approach described here adds to the arsenal of endoscopic management techniques for presacral sinuses that often develop after IPAA surgery. This spares patients from the invasiveness and high risk of morbidity associated with the conventional surgical interventions. To our knowledge, this is the first case of successful CT-guided localization of a presacral sinus with a percutaneous guide wire to allow for endoscopic NKSi.Conflict of interest statement: The authors declared no financial conflicts of interest. Dr. Bo Shen is supported by the Ed and Joey Story Endowed Chair.


The inclusion for rehabilitation of visually impaired children is partly based on the measurement of near vision, but guidelines for near visual acuity assessment are currently lacking. The twofold purpose of this systematic review was to: (i) provide an overview of the impact of the chart design on near visual acuity measured, and (ii) determine the method of choice for near vision assessments in children of different developmental ages.A literature search was conducted by using the following electronic databases: PubMed, Cochrane Library, and EMBASE. The last search was run on March 26th 2016. Additional studies were identified by contacting experts and searching for relevant articles in reference lists of included studies. Search terms were: vision test(s), vision assessment(s), visual acuity, chart(s) and near.For children aged 0–3 years the golden standard is still the preferential looking procedure. Norms are available for this procedure for 6–36 month old children. For 4–7 year olds, we recommend using the LEA symbols, because these symbols have been properly validated and can be used in preliterate children. Responses can be verbal or by matching the target symbol. In children aged 8–13 years, the recommended method is the ETDRS letter chart, because letter acuity is more predictive for functional vision and reading than symbol acuity. In 8–13 year olds, letter acuity is 0.1–0.2 logMAR poorer than symbol acuity.Chart design, viewing distance, and threshold choice have a serious impact on near visual acuity measurements. Near visual acuity measured with symbols is lower than near visual acuity measured with gratings, and near visual acuity measured with letters is lower than near visual acuity measured with symbols. Viewing distance, chart used, and letter spacing should be adapted to the child’s development and reported in order to allow comparisons between measurements. Near visual acuity measurements are an important part of general vision assessment. Our ability to see small details up close can be measured with near vision charts. One problem in near visual acuity assessment is that there are numerous charts available and the number is consistently growing. The measurement of near visual acuity in children is not yet part of the routine ophthalmological examination. It is time consuming and requires special skills because children are eager to shorten the viewing distance which complicates measurement of near visual acuity. Sensitivity between near visual acuity charts varies largely and has a major impact on measurement of visual acuity. It is important that clinicians are aware of these differences. The inclusion for rehabilitation of children with visual impairment (VI) is based on their near visual acuity. The outcome of near visual acuity assessment thus has personal as well as economic consequences. There is broad consensus among researchers that chart design and reading distance have an impact on near visual acuity scores, but a systematic overview of the results collected with different near vision charts is currently lacking. Reproducibility of near visual acuity measurements depends on the age and co-operation of the child and the method used. The choice for a method should be based on the developmental level of the child. This systematic review will provide guidelines for near visual acuity measurements for 0-3 year old children with normal visual acuity and low vision.The measurement of near visual acuity is dependent on a number of factors, such as distance visual acuity, accommodation capability, and near vision correction . The ampNear visual acuity can be measured in different ways, of which the following two are most often used in clinic: i) resolution acuity, for example tasks where the stimulus pattern (grating) has to be resolved or chosen or a gap has to be detected, and (ii) recognition acuity, for example Snellen letters or LEA symbols . We willThree databases were used to select studies: PubMed, Cochrane and EMBASE. Quality of the included studies was evaluated independently by two reviewers (BH and FNB) using criteria for cross sectional studies. The last search was run on March 26th 2016. Studies that reported near visual acuity outcome measures were included if they met four inclusion criteria: (1) a cross-sectional or observational design was used, (2) the study included 0–13 year old children with normal development and normal vision, and/or 0–13 year old children with normal development and low vision, (3) the study included children without mental impairments, and (4) the study was published in an English peer-reviewed journal. The search was developed by an experienced clinical librarian together with the first author of this article. After selecting all possible studies with predefined search terms (see search strategy in Table Study eligibility based on inspection of titles and abstracts was performed by the two authors (BH and FNB), using the inclusion criteria presented in Table Included quantitative studies had to adhere to the following criteria: 1) report near visual acuity scores in one of our two target groups, 2) (lowest inclusion) age of children should be between 0–13 years, and 3) the study has a cross sectional or observational design. In order to increase data collection we also searched for articles by inspecting reference lists of the included studies. Screening studies were included, but studies only involving children with amblyopia were excluded from this review since the focus is explicitly on children with normal vision and children with low vision. Children with refractive errors were included, since they can attain normal visual acuity when corrected properly.Quality of the included studies was evaluated independently by two reviewers (BH and FNB) using criteria for cross sectional and case-control studies. Information for evaluation of the included studies was: number of participants, method used, a clear outcome definition , and results . The first author extracted the data and contacted authors of identified studies for additional data if necessary.Because of the scarcity of studies providing quantitative data about near vision in children and the wide range of vision tests used to measure near vision we report results in a narrative rather than a quantitative manner.n = 40), they were not published as an article (n = 6), or because they included the wrong population (n = 9). After doing this, 34 studies remained. Of the included studies, 29 were quantitative studies, and 5 were qualitative studies. See PRISMA flow chart Fig. Our search in the three electronic databases provided a total of 602 citations. After removal of duplicates there were 436 studies left. After screening these studies on their titles and abstracts the number of studies for inclusion was 89. The next step was to screen full-text versions of these articles for eligibility. Fifty-five studies were excluded because they did not contain the primary outcome measure  detection of the optokinetic nystagmus (OKN), and (ii) the use of preferential looking techniques . The OKNAnother method that can be used to measure visual acuity in infants is preferential looking. Preferential looking is a procedure based on the observation that infants have a greater tendency to fixate a pattern than a homogeneous field. Position and location of the stripes is varied on each trial and acuity is, like for the OKN, expressed as the smallest stripe width the infant can see. Among preferential looking measurements there was also a good amount of agreement across research groups with regards to the mean acuity of infants; values fell within one octave of each other. In the ‘80s there was already a trend for preferential looking to be used more frequently than OKN for laboratory measurements. Factors favouring preferential looking above the OKN method are: stimuli are easier to produce, and forced choice preferential looking (FPL) is less subjective than OKN. Judging the presence of an OKN can be difficult and in FPL proportions correct are scored and a psychometric curve can be plotted. Dobson provides three criteria for tests to be clinically useful: efficiency (measurements should not take too long), facility (tests should be easy to adopt), and validity (infants with poor acuity should do poorly on the test).The second qualitative review was written 3 years later by Davida Teller . Teller In addition to the review studies, 10 quantitative studies were found, spanning a total time period of 51 years. One study measured visual acuity in newborns by detecting an OKN . The othTable The golden standard for testing near visual acuity in 0–36 month old children is the TAC, providing norm scores across the whole age range and validated for children with NV  and low More than 30 years after Teller’s review, there are now developments towards a computerized version of the TAC using eye-trackers to measure gaze direction. This new method offers a more objective and standardized way of measuring visual acuity in children than the TAC, because TAC acuities rely solely on a subjective report of whether or not the child’s gaze was directed at a target. A computerized version of the TAC has several advantages: (i) it is fully automated and scoring does not rely on an observer, and (ii) it has control over key parameters that, ideally, should be standardized . However, it also has disadvantages, because certain eyes are not easy to track, for instance because of nystagmus or iris transillumination. So, a computerized application of the TAC might be very useful, but since eye-tracking is not always an option, it is likely that TAC cards will still be used in the future.A total of 12 studies were included –32. ThreChildren aged 3 years and older can reply in a verbal manner and can grasp the concept of matching an answer to a stimulus. The response manner in the majority of studies is verbal naming or matching. Thirty years ago, a review on vision testing in 3–5 year old children reported that matching works reliably in a number of studies, while young children may refuse to give an adequate verbal response to testing . The numAs can be seen in Fig. In children aged 4–7 years, near visual acuity can be measured with crowded and uncrowded acuity charts. In three of the included papers uncrowded and crowded visual acuities were compared , 27, 28.As can be seen in Table In children aged 4–7 years there is not such a clear golden standard as there is in 0–3 year olds. Near visual acuity can be assessed with several validated tests with comparable outcomes, for example the crowded Landolt C-test , the croTen studies were included in which near visual acuity was assessed in children aged 8 years and older , 37–45. In the 70’s and 80’s the Sheridan Gardener test was often used to measure near visual acuity in school aged children and the Snellen chart was used to measure distance visual acuity , 43. NowTwo studies were included on near visual acuity assessments in subjects with VI , 45. TheThe second study in which near visual acuity was measured in children and adults with VI due to infantile nystagmus aimed to assess differences in near and distance visual acuity . This waThe ETDRS was the most frequently used chart to measure near vision in children aged 8 years or older. There are clinically relevant differences in visual acuity measured with letters and visual acuity measured with symbols (differences appear to be ~0.1–0.2 logMAR). Therefore, clinicians should report the chart that was used to measure acuity. The relatively lower acuity for ETDRS letter charts might be caused by a relatively low guess rate (10%), and the use of SLOAN letters, which are more difficult to discriminate than resolution or symbol optotypes. The benefit of using letters instead of symbols is that letter recognition lies closer to reading ability than symbol recognition.The general goal of this review was to provide guidelines for near visual acuity measurement for 0–13 year old children with normal vision and low vision. The most frequently used method to measure visual acuity in 0–36 month old children was the TAC. There was no clear method of choice for the intermediate age group, the 4–7 year old children. For the 8–13 year old children, the ETDRS was the most frequently used chart. Nine studies were included that measured near and distance visual acuities (reporting means and standard deviations), but there were no systematic difference between these measures. Standard deviations were not larger for near than distance visual acuities, suggesting that reliability of these measures is not weaker than reliability of distance visual acuities when measured with a standardized routine. This review brings forth several insights with regards to four topics: (i) the impact of threshold choice on vision outcome, (ii) the sensitivity of charts to detect refraction errors or crowding, (iii) the impact of viewing distance during near vision assessments, and (iv) the impact of optotype choice on the measured visual acuity.For 0–36 month old children, the TAC procedure is the clear golden standard. During the development of the TAC in the early ‘80s, there were studies that compared outcomes collected with different thresholds  . An explOptotype choice has an impact on near visual acuities measured with letter scores resulting in lower acuity estimates than charts with symbols. This difference between acuity measured with letters and acuity measured with symbols was consistent across several studies and several age groups. Visual acuities measured with the 4AFC C-test and LEA symbols correspond well with each other , 28. TheThere were no consistent differences between distance and near visual acuities. Myers et al. did not find a difference between near and distance ETDRS acuity in children with normal vision . LarssonThis review shows that for 0–36 month old children there is a golden standard for near visual acuity assessments: the Teller Acuity Cards (TAC). In 4–7 year olds, we recommend use of the LEA-chart at 40 cm because it has been validated properly and can be used in preliterate children without inducing left-right confusion. In 8–13 year olds, the ETDRS seems to be the preferable chart. The following guidelines for clinical practice can be extracted from this review: (i) visual acuity should be measured with the most sensitive chart that is validated for a variety of subjects, (ii) the choice for a specific chart should be based on developmental age, (iii) different norms should be used for crowded and uncrowded acuities and norm values applied to estimate the age-dependant amount of crowding, (iv) for very young children, low thresholds or maximum correct response should be used, and (v) viewing distance and thresholds should be standardized (preferably a viewing distance of 40 cm).


While these were initially the product of public health agencies and health-related NGOs, they are increasingly being developed and disseminated by the alcohol industry. There is considerable debate as to whether industry-generated campaigns are designed to reduce hazardous drinking and related problems (as argued by their developers) or are designed to avoid government regulation or even to increase sales. The aim of the present study was to explore the way that recent industry-developed responsible drinking campaigns are perceived and interpreted by the general public. That is, do they promote low-risk drinking, promote risky drinking, or just muddy the waters. Two sub-studies were conducted. The first, a mall intercept study with 180 adults in two Australian shopping districts, explored participants’ understanding of slogans/taglines. The second, an online survey with 480 Australian adults, explored understandings and interpretations of television/online commercials. The results of the two studies revealed diversity in participants’ interpretation of the ‘responsible drinking’ advertisements. Terminology utilised in industry-developed advertisements was found to be ambiguous; for example, what age group was being referred to in the tagline ‘Kids and alcohol don’t mix’, and whether ‘Drink Properly’ meant not drinking to excess or drinking in a way that made you look more sophisticated. In Study Two, the government-developed campaign (‘Know when to say when’) was clearly interpreted as warning against risky consumption of alcohol; whereas the industry-developed campaigns  were interpreted to have a range of different meanings, including some seemingly unrelated to alcohol. These findings are consistent with the literature evaluating anti-smoking campaigns developed by the tobacco industry, and previous research showing that industry communications serve to soften public opinion and create the impression of a ‘socially responsible’ industry but are likely to be less effective than initiatives focused on the availability and promotion of alcohol. According to the Australian Bureau of Statistics, there were 9.7 litres of pure alcohol available for consumption per person in 2013–14 ;‘Know when to say when’ [from a NSW Government education initiative];‘You won’t miss a moment if you DrinkWise’ [DrinkWise];‘Drinking: Do it properly’ [DrinkWise];‘Kids absorb your drinking’ [DrinkWise]; and‘Kids and alcohol don’t mix’ [DrinkWise].For each slogan/tagline, respondents were asked an open-ended question: “What does the following statement mean to you?”They were then asked to identify the age group(s) referred to in the message ‘Kids and alcohol don’t mix’ . Given the debate regarding the purpose of the Drinkwise ad ‘How to drink properly’ that was occurring at the time [The survey instrument was pre-tested using one-on-one cognitive interviews with a convenience sample of 6 men and 13 women, 18 years of age and older, recruited from the researchers’ networks via email or phone . This alParticipants were recruited using a combination of advertising on social networking sites (n = 177) and a commercial recruitment agency to ensure our age and gender quotas were met (n = 310). A total of 487 people completed the online survey; seven participants were excluded because their responses were incomplete or clearly vexatious, leaving a sample of 480 for analysis.Survey Monkey 2013 was used as the survey platform. The online survey was pre-tested with the same convenience sample that pre-tested the intercept survey (6 men and 13 women aged 18 years and older). The average time to complete the online survey was 15 minutes. Minor revisions were made following pre-testing.Demographic questions were asked immediately after completing the consent process, to enable the screening out of ineligible or over-quota respondents. Four video (TV or online) advertisements were then shown in random order see . FollowiDescriptive analyses were computed for all demographic variables for Study One and Study Two participants.For both Study One and Two, categorical data were entered into the Statistical Package for the Social Sciences (SPSS 22.0) and descriptive analyses were conducted.The deductive coding of open-ended responses involved an iterative process whereby two independent coders (SJ and SH) reviewed the responses to Survey One and developed a list of codes; and then coded all responses against these codes. Initial poor levels of agreement indicated a need to further refine the coding guide. Thus two separate coders (JL and KK) reviewed a subset of the responses  and coded them independently. Following discussion between the four coders the coding guide was revised with clear explanation of the meaning and inclusions of each code. The lead author (SJ) then re-trained one of the initial coders (SH) and an additional coder (JT) who independently coded all of the open-ended responses. The inter-rater agreement across the two reviewers was 99.4% for the intercept survey and 99.7% for the online survey. Discrepancies were resolved through group discussion with the lead author as arbiter.Consistent with the quotas established, 90 respondents were recruited in each of Melbourne and Newcastle (45 men and 45 women in each location). Approximately one-third were aged 16–25 years (n = 58), 26–45 years (n = 62) and 46 years and over (n = 60). Additional demographic information on the sample is provided in Across the four taglines targeting adult drinking  17 unique interpretations were coded. This excludes those who simply stated that they agreed or disagreed with the message (between 11 and 22 across the four ads), gave a response that was too vague to code (n = 25 to 49), or did not provide a response (n = 6 to 10). These 17 codes were reduced to 10 over-arching codes , thinking about what you are drinking , knowing your limits  and not drinking to drunkenness . About half of the respondents (n = 108) identified that ‘Know when to say when’ was about knowing your limits and not drinking beyond those limits; this response was more common among females (66%) than males (54%). No other interpretation was identified by more than 6% of respondents.A wide range of meanings were proposed for the tagline The most common interpretation of ‘You won’t miss a moment if you DrinkWise’ was avoiding drunkenness . It is noteworthy that 20.6% (n = 37) specifically identified the message as being able to remember things after a night of drinking/not having blackouts; this response was more common among females (26%) than males (16%). Other messages identified were drinking in moderation  and still being able to have fun without excessive drinking .The most common interpretation of ‘How to drink properly’ was drinking in moderation . Other interpretations identified by more than 20% of respondents were avoiding drunkenness  and thinking about what you are drinking . Fifteen respondents (8%) interpreted this message to be a call to drink more rather than less alcohol.2 =χ 9.27, p = 0.002); drinking the right kind of alcohol ; and knowing how to mix a drink .Given the debate referred to above, it is important to understand how respondents interpreted the term “properly”. Of the seven options presented to respondents see , the mosEleven unique interpretations were coded across the four taglines targeting parents and other adults’ influence on children’s drinking; excluding those who simply stated that they disagreed with the message in either of the two ads (8 and 3 respectively), gave a response that was too vague to code (14 and 16 respectively), reported that they did not know what the message was (5 and 2 respectively), or did not provide a response (6 and 2 respectively). These 11 codes were reduced to seven over-arching codes  thought that the tagline ‘Kids absorb your drinking’ meant that children imitate their parents’ behaviour, with 22% (n = 39) making a clear connection to alcohol (e.g. ‘kids emulate your drinking habits’); this latter interpretation was considerably more common among female (28%) than male (16%) respondents. No other interpretation was provided by more than 8% of respondents.Almost half (n = 80) of the respondents indicated that ‘Kids and alcohol don't mix’ meant that children should not drink, or be provided with, alcohol. The next most common response  was a statement of agreement with the message, although it is unclear what these respondents perceived that message to be. Approximately 12% perceived the message to be that they should drink responsibly in front of children (n = 22) and a similar number that they shouldn’t drink in front of children (n = 20).Given that almost half of the respondents interpreted this last slogan to mean that they should not provide children with alcohol, it was important to know what they believed the word ‘children’ referred to. In response to that question, only 54% agreed that ‘kids’ in ‘kids and alcohol don’t mix’ also included 16–17 year olds; whereas 81% believed it also included 13–15 year olds and 75% that it also included 7–12 year olds.Consistent with the quotas established, approximately one-third of the 480 respondents were from Victoria (n = 156), New South Wales (n = 177) and the rest of Australia (n = 147). Approximately half were male , with a fairly even split across the three age groups of 16–25 years (n = 140), 26–45 years (n = 159) and 46 years and over (n = 181). Additional demographic information on the sample is provided in Twenty-two unique interpretations were coded across the three taglines; again this excludes those who simply stated that they agreed or disagreed with the message (between 3 and 6 across the three ads), gave a response that was too vague to code (n = 1 to 7), or did not provide a response (n = 9 to 16). These 22 codes were reduced to 11 over-arching codes  and those without a tertiary education . Other common interpretations were not becoming drunk , avoiding negative outcomes  and drinking in moderation .The predominant interpretation of the ‘Know when to say when’ advertisement was knowing your limits . This was more common among females , those aged less than 25 , and those without a tertiary education . Other commonly noted interpretations were drinking in moderation , not becoming drunk , and avoiding negative outcomes –both of the latter showed a particular emphasis on not embarrassing yourself. Nineteen respondents commented that the main message was about being sophisticated and 16 that it was to drink more.Two interpretations were prominent in response to the ‘How to drink properly’ advertisement. The most common of these was knowing your limits , including references to previous experience and knowing how much alcohol you can handle . This wa2 = 4.72, p = 0.03). A smaller proportion mentioned drinking in moderation ; thinking about what you are drinking ; and avoiding negative outcomes, with a particular focus on harm to others . Those aged 25 or less were more likely to note the message as thinking about what you are drinking  and avoiding negative outcomes , and less likely to perceive the message as to drink in moderation . Those with as tertiary education were more likely to perceive the message as drink in moderation  and less likely to see it as avoiding negative outcomes .The predominant interpretation of the ‘Friends are waiting’ advertisement was don't drink and drive . This was followed by friendship , with half of these  mentioning alcohol  and the remainder not specifically referring to alcohol . Women were more likely than men to note friendship as the message , gave a response that was too vague to code (n = 25), or did not provide a response (n = 12). These nine codes were reduced to seven over-arching codes see . The mosThe primary perceived target group for the ‘How to drink properly’ and ‘Friends are waiting’ campaigns was young adults aged 18–25 years; whereas for the ‘Know when to say when’ campaign it was adults aged 25 years and over. Parents were seen as the main target audience for ‘Kids absorb your drinking’.2χ = 4.061, p = 0.04).The advertisement participants were most likely to report was relevant to them was the US ‘Friends are waiting’ (42%), followed by ‘Know when to say when’ (37%), ‘How to drink properly’ (35%) and ‘Kids absorb your drinking’ (33%). Conversely, the ad perceived as most likely to change their behaviour was ‘Kids absorb your drinking’ (40%) and the ad least likely to impact on their drinking was ‘How to drink properly’ (23%). The only gender difference was that female participants were more likely to perceive ‘Friends are waiting’ as relevant to them  was clearly interpreted as warning against harmful consumption of alcohol. However, both of the SAPRO-developed and industry-developed adult-targeted campaigns  were interpreted to have a range of different meanings–including some seemingly unrelated to alcohol  and some seemingly encouraging or glamourising drinking .The findings are consistent with previous research showing that industry communications about their products are self-serving, sometimes in ways that are not immediately obvious . First, There were noteworthy differences in interpretation by level of educational attainment of the respondents. For both the ‘Know when to say when’ and ‘How to drink properly’ advertisements, those without a tertiary education were more likely to interpret the message as knowing your limits. This refers to a subjective assessment of a safe or appropriate level of alcohol consumption based on previous experience and knowing how much alcohol you can ‘handle’, rather than an objective assessment of a quantity of alcohol based on medical or health guidelines. Thus, it may be particularly important to develop messages for this group that communicate the unreliability of this assessment for avoiding short- and long-term harms from alcohol consumption. Conversely, those with a tertiary education were more likely to interpret the ‘drink properly’ slogan as a call to appear more sophisticated when drinking, for example to drink the right kind of alcohol and know how to mix a drink. Campaigns targeting this group may need to focus on countering perceptions of drinking as glamorous and indicative of social success or status.Limitations of the study include the reliance on participant introspection and attribution as a basis for inference as to the meanings of the advertisements. However, we would argue that it is viewers’ interpretations of these meanings that are of primary importance, rather than the stated intentions of the advertiser. While a viewer’s impressions of advertisements are, by definition, subjective, it is possible that their behaviour, e.g., how they drink, is weakly correlated with the verbal or web-based responses given in a survey. A research context provides artificial conditions for the viewing of advertisements, and it is uncertain how this affects inferences. It may be expected that participants answer questions in ways they think might be expected from a university researcher, a phenomenon referred to as ‘research participation effects’ . HoweverStrengths of the research include the use of two sampling approaches (street intercept and survey panel), and survey modalities (face-to-face and web), and different locations (Melbourne and Newcastle), suggesting that the findings, which were similar across the studies, are robust.The evidence for reducing the population’s exposure to the marketing activities of corporations that sell dangerous products such as alcohol is clear. However, despite substantial levels of public support for greater restrictions, successive governments in Australia–as in many other countries–have been reluctant to do so. The challenge is in securing changes in policy in the face of strong industry resistance to regulation, and skilled lobbying for the status quo or relaxation of existing restrictions.We found that participants clearly understood the message of the government campaign as a warning against harmful drinking, but many found the SAPRO-developed campaigns to be ambiguous. This study adds to a small but consistent body of research in the Australian context that the activities of SAPROs, and other alcohol industry initiatives, serve to soften public opinion and create the impression of a ‘socially responsible’ industry while being ineffective in moderating consumption. There would be value in investigating policy makers’ perceptions of such initiatives and the role of SAPROs and industry lobbying in avoiding evidence-based regulatory changes to reduce alcohol-related harms.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S3 Appendix(DOCX)Click here for additional data file.S4 Appendix(DOCX)Click here for additional data file.S5 Appendix(DOCX)Click here for additional data file.S6 Appendix(RTF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.S2 Dataset(XLS)Click here for additional data file.


Many people begin to consume alcohol and establish drinking patterns during adolescence, making this an important developmental period for alcohol researchers to study. Both drinking initiation and establishment of drinking patterns are influenced to varying degrees by genetic as well as environmental factors. Using twin studies conducted in Finland and other countries, researchers have analyzed the specific genetic and environmental influences as well as the gene–environment interactions that shape drinking behavior in adolescence. These studies indicate that drinking initiation is determined primarily by environmental influences, whereas the establishment of drinking patterns is determined mostly by genetic factors, which themselves are subject to moderation by the environment. The years between early adolescence and young adulthood are a crucial period for alcohol researchers to study, because drinking typically is initiated during adolescence, and by young adulthood, individual differences in established drinking patterns are evident. The earliest stages of alcohol use merit particular attention because research has suggested that the initiation of alcohol use must be distinguished from the frequency, quantity, and density  of alcohol use once drinking is initiated. Consequently, analyses in adults with already established drinking patterns can explain only part of the mechanisms and contributing factors underlying the development of alcohol abuse and dependence. To obtain a more comprehensive explanation of these processes, it also is necessary to study the factors influencing drinking initiation.Another reason to focus on early adolescent drinking comes from research evidence indicating that people who begin drinking at an earlier age have a heightened risk for negative consequences of drinking, such as alcohol abuse or alcohol dependence. For example, interviews of adults consistently and convincingly have shown a strong association between an early initiation of drinking and later alcohol-related problems: People who report drinking as early as age 13 or 14 are much more likely to exhibit alcohol-related behavior problems in adulthood than people who began drinking at a later age . The assBut what is the relevance of this association? In most cases, it is hazardous to draw a causal connection based on such observations. Thus, it is possible that drinking initiation at an earlier age is causally related to greater vulnerability to alcohol’s cumulative effects. Alternatively, early onset of drinking and a greater likelihood of later problems may simply be correlated—that is, they are independent signs or symptoms of people who are at elevated risk of alcohol dependence. In the latter case, prevention efforts designed to promote delays in age of drinking initiation would do little to reduce the prevalence of adult alcoholism—an issue that is important to resolve. One approach to doing this is to conduct longitudinal studies that follow, over several years, genetically informative samples, such as samples of twins who differ in their age at onset of drinking.FinnTwin studies help investigators address questions about factors contributing to various stages of alcohol use. The resulting research has underscored the importance of the concept of dynamic gene–environment interactions.This article reviews recent findings from two ongoing longitudinal twin studies conducted in Finland that elucidate the contributions of genetic and environmental influences to initiation of drinking and later trajectories of alcohol use and abuse. Data from these population-based Both genetic and environmental factors contribute to the different stages in a person’s drinking history. The factors that influence individual differences in whether, and at what age, drinking is initiated are different in kind from the factors that influence individual variations in patterns of drinking, once the acute subjective effects of alcohol ingestion are experienced. Moreover, it is clear from numerous studies that although genetic factors have some influence on drinking behavior, the effects of genetic differences between people can be enhanced or reduced by variations in the environments in which the people live—that is, there are gene–environment interactions.A person’s susceptibility to developing alcohol dependence and other alcohol-related problems manifests as an unfolding developmental process that involves both genetic and environmental factors and is continuously modulated by gene–environment interplay. This concept of a complex developmental process is supported by fundamental findings in recent behavior-genetic research that have profoundly altered the earlier view that people are passive players who mechanically mirror their environmental experience. Instead, recent research findings indicate the existence of extensive gene–environment correlations, whereby people are active agents who help create the experiences to which they are exposed, selectively seek environments to match their specific dispositions, and modify their environments through their actions. In short, new research suggests that constant gene-by-environment interplay is involved in the development of alcoholism risk over time.To understand the factors affecting the development of alcoholism, it is essential to appreciate this continuing interaction of genetic and environmental influences . Similarly, it is important to acknowledge that the household environment does not stand alone, but is “nested” within neighborhoods, schools, communities, and cultures. Accordingly, the ways in which a child expresses his or her internal dispositions are modulated by the adult models, parenting practices, peer interactions, access to alcohol, and school and neighborhood environments to which the child is exposed. All of these factors either enhance or reduce the risk for alcoholism that results from the child’s genetic makeup.FinnTwin studies.Longitudinal studies using genetically informative samples enable researchers to assess the magnitude of genetic and environmental influences and their interplay across development for a variety of behaviors. Therefore, these research designs also are particularly useful in understanding the development of drinking behaviors. The most widely used design is a longitudinal/developmental study of child or adolescent twins and their parents. Several research groups in the United States and other countries are engaged in developmental behavior-genetic research of this kind, and many of these groups explore risk factors for alcoholism as part of their research aims. This article focuses on two longitudinal studies of young Finnish twins and their parents known as the Finland is particularly suitable for conducting longitudinal twin studies because of the availability of comprehensive birth records, the ease of locating twins as they grow up, and the high level of compliance of organizations and individuals with research requests.To acquire a large sample of twins that is representative of the entire population, it is almost imperative that investigators have access to a comprehensive birth registry for the region or country under investigation. However, many countries, including the United States, do not have such registries. Some researchers in the United States have attempted to obtain comprehensive data sets by identifying all twin students enrolled in a large public school system or by systematically accessing birth records within a State and then searching for the current addresses of the twins so identified.In the Nordic countries, in contrast, the entire population is enrolled at birth into a centralized population registry. In Finland, every newborn citizen is given a unique personal identification number that includes the person’s date of birth and a link to the biological mother. Accordingly, researchers who have approval to access the registry can identify all people born on the same day to the same mother, thereby identifying the nation’s entire population of twins and other multiples. Moreover, Finnish law requires all citizens to report all residential moves in a timely manner, which means that families of twins can be readily located and, in principle, followed up without loss. Finally, for cultural and historic reasons, compliance with requests for research participation is unusually high in the Nordic countries. This high compliance rate and the requirement to report residential moves make it possible to conduct followup assessments with large numbers of subjects, which otherwise presents a particular challenge when researching drinking behavior and its consequences .FinnTwin studies differ in the age groups they study and in their followup intervals. One study, Finn-Twin16–25, includes all twins born in five consecutive birth cohorts, 1975 through 1979, who were enrolled in the study when they reached age 16. Systematic followup surveys of these twins were completed at ages 17 and 18.5 and at one point when the twins were between ages 22 and 25. The second study, FinnTwin12–17, identified all twins in the five consecutive birth cohorts 1983 through 1987 who were enrolled at age 11 or 12, with followup assessments at ages 14 and 17.The two FinnTwin study includes about 2,500 pairs of twins, with equal proportions of brother–sister, brother–brother, and sister–sister twin pairs. Among the same-sex pairs, about half are identical, or monozygotic (MZ) twins and half are fraternal, or dizygotic (DZ) twins. MZ twins develop when a single egg is fertilized by a single sperm, and the resulting embryo divides shortly after fertilization to produce two individuals who share all their genes. DZ twins, in contrast, develop when two egg cells are independently fertilized by separate sperm at about the same time , resulting in the development of two individuals who, like ordinary siblings, share, on average, half of their genes.Each The fundamental approach in twin study research is to compare the extent to which MZ twins resemble each other in relation to a specific trait or behavior with the resemblance between DZ twins for the same trait or behavior. In MZ twins, the genetic influences on that trait or behavior are identical because the co-twins share all their genes. In DZ twins, however, the genetic influences can differ because the co-twins share only about 50 percent of their genes. With respect to environmental influences, there should be no substantial differences between MZ and DZ twins because, in childhood and adolescence, all or nearly all twin pairs live with one another and with the same biological parent or parents. Therefore, both MZ and DZ twins will share equally important aspects of their household experience, peers, neighborhood, school, and other environmental factors that may enhance or reduce the risk for early alcohol use and abuse.Based on these premises regarding genetic and environmental influences, the following expectations can be articulated:If genetic factors are primarily important in the development of a measured trait or behavior, MZ co-twins will resemble one another about twice as much as do DZ co-twins.If genetic factors are of only modest importance and shared environmental influences are predominant, the two kinds of twins will show comparable resemblance for the trait or behavior studied, because both share the environmental influences relevant to that outcome.FinnTwin studies regarding the contributions of various genetic and environmental factors to a variety of drinking-related behaviors. In general, the results from the FinnTwin studies have been consistent with ongoing research with adolescent twins in other European countries as well as in the United States.The following sections summarize some of the findings of the FinnTwin12–17, which addressed this issue. Nearly two-thirds (64 percent) of the twins in this study reported never having used alcohol at age 14, whereas 36 percent reported that they had begun drinking alcohol at this age. Comparisons of the MZ and DZ twin pairs in the study determined that the environmental factors shared by co-twins played the largest role in influencing abstinence/drinking at age 14. These factors—which included the familial environment and the nonfamilial environments of peers, schools, and neighborhoods—accounted for 76 percent of the variation in drinking initiation in both boys and girls . Thus, although many environmental influences impacting drinking initiation are the same for girls and boys, some unidentified environmental influences are specific to each gender.One relevant question in this context is whether the same factors influence drinking initiation in 14-year-old boys and girls. Although the FinnTwin data provided little evidence of genetic effects on drinking initiation by age 14, some gender differences were apparent in that respect as well. Genetic effects were found to have a modest influence on girls (accounting for 18 percent of the variance in girls’ drinking at age 14) but no influence on boys’ drinking at the same age. Perhaps this difference reflects the fact that girls are more developmentally mature at age 14 than boys. By this age, most girls already have undergone significant pubertal development, whereas most boys are just beginning to experience pubertal changes. Girls’ more advanced maturation may foster associations with older friends and peers that offer greater access to alcohol, thereby creating opportunities for girls to express drinking-related genetic predispositions at an earlier age.Although the FinnTwin 12–17 study. Only 12.4 percent of the 14-year-old twins in this sample reported any symptoms of alcohol dependence, and only 1 percent met the criteria for a diagnosis of alcohol dependence. Moreover, more girls than boys reported alcohol dependence symptoms or met diagnostic criteria; this gender difference may be attributable to the fact that, at this age, girls are more likely than boys to be drinking.It is uncommon for 14-year-olds to exhibit symptoms of alcohol dependence, and at this age such symptoms are not attributable to genetic effects. To explore the issue of alcohol dependence at this early age in more detail, researchers administered a structured face-to-face interview to a sample of more than 1,800 twins from the For comparison, the investigators also determined the frequency of symptoms of conduct disorder (CD) in the same sample of twins. This analysis yielded several results that differed sharply from those obtained regarding alcohol dependence symptoms :Symptoms of CD were common among the adolescent twins: 44 percent of the sample reported one or more symptoms of CD, and 12 percent met diagnostic criteria for the condition.CD symptoms were more common among boys, who accounted for 65 percent of all twins diagnosed with CD.Genetic factors played a much different role in CD than in alcohol dependence. Whereas genetic factors had a negligible influence on alcohol dependence, they had a substantial influence on symptoms of CD.Despite these differences, the two sets of symptoms are significantly correlated (0.50), meaning that the CD symptoms were more common among twins with a diagnosis of alcohol dependence and, similarly, the symptoms of alcohol dependence were more common among twins diagnosed with CD. However, because the twin data produced no evidence that genetic factors contribute to alcohol dependence symptoms at this age, the association of alcohol symptoms with CD symptoms must be attributed entirely to environmental factors that influence both conditions . One infFinnTwin, include not only household environmental characteristics  but also environmental influences exerted through schools, neighborhoods, and communities. For some behaviors and at some ages, the nonfamilial experiences that children share with their peers at school and in their neighborhoods may be more salient than the household influences they share with their families in their homes. Drinking in early adolescence may be one such behavior. To evaluate this hypothesis, researchers can assess the contribution of nonfamilial environments to children’s behavior by comparing the resemblance of unrelated classmates of twin siblings to one another and to their classmate twins.The environmental factors shared by siblings, and assessed in twin studies such as FinnTwin12–17 study undertook such an approach  must result entirely from experiences shared in school and neighborhood. Accordingly, this research approach provides a direct estimate of the role of environmental influences associated with schools and neighborhoods.FinnTwin12–17 study, behavior of the twins and the classmate control subjects was assessed when the children were 11 or 12 years old by means of a questionnaire administered during school hours under research supervision. The questionnaire included three drinking-related items illustrating nonfamilial environmental effects:In the “Have you ever drunk alcohol?”“Have you ever seen any of your friends drunk?”“Have you ever drunk alcohol with your friends without adults around?”The prevalence of affirmative answers to these questions differed widely, as would be expected among children at this age. For example, more than two-thirds of the boys and more than half of the girls admitted to having had a drink of alcohol—which is not uncommon in Finnish family culture, where children may have a drink at holiday time and with parental knowledge. In contrast, only 18 percent of boys and 11 percent of girls reported having seen friends drunk, and only 7 percent of the boys and girls reported ever having drunk alcohol with their friends without adults around.The investigators determined the relative influences of genetic and specific environmental factors on these three variables based on the relative similarities of MZ and DZ twins and their classmate control subjects see . They foThus, this expanded twin–classmate control design has identified important influences on drinking behavior that originate both within and outside the family environment. These findings document the varying influences of a person’s genetic dispositions on different aspects of drinking behavior in early adolescence.FinnTwin 16–25 study illustrate this process.The relative contributions of genetic and environmental factors to the drinking behavior of young people change during adolescence. As previously described, familial and environmental influences predominate in their impact on initiation of drinking behavior. When people move from early experimental alcohol use to more established patterns of use, however, the importance of individual genetic predispositions increases greatly, and the importance of common environmental factors decreases accordingly. Findings from the FinnTwin16–25 was an assessment of the twins at ages 16, 17, and 18.5. Over this 30-month period of followup, the number of abstinent participants decreased dramatically, from about 25 percent of twins at age 16 to only about 10 percent of twins at age 18.5. Conversely, the proportion of twins who reported drinking at least twice monthly more than doubled over the 30-month period, and at age 18.5 more than 60 percent of twin boys and girls drank that often  now make it possible to move beyond estimates of unmeasured  sources of genetic variance. Researchers have begun to identify specific genes involved in complex behavioral outcomes such as alcohol use.Using a candidate gene approach, which examines the influence of specific genes suspected of playing a role in the behavior under investigation, two studies recently reported associations between specific gene variants and drinking behavior among college students. The first study investigated the role of variation in the gene encoding a protein called the serotonin transporter. This study found that in a sample of White students, those with a particular version of this gene engaged in binge drinking more often, drank to intoxication more often, and consumed more alcoholic drinks per drinking occasion than students with other variants of the gene .Another study focused on the gene that encodes aldehyde dehydrogenase (ALDH), an enzyme involved in the breakdown of alcohol in the body. This study reported that Asian American college students who carried a particular version of the ALDH gene which results in less efficient alcohol breakdown were less likely to be regular drinkers and engage in binge-drinking episodes; they also reported a lower number of maximum drinks consumed in a 24-hour period than Asian students with other ALDH variants .Such studies are complemented by large-scale efforts to identify genes that contribute to alcoholism. One of these efforts, funded by the National Institute on Alcohol Abuse and Alcoholism, is the Collaborative Study on the Genetics of Alcoholism (COGA). COGA researchers recently published reports of several genes associated with alcohol dependence in adults , and somThe next challenge will be to expand these findings to children and adolescents to better understand how specific predisposing genes influence trajectories of drinking and other behavioral problems across development. The identification of specific genes influencing alcohol use and abuse will enable researchers to begin to decipher how the effects of these genes are modified by specific environmental influences. Elucidating the influence of specific genes on various aspects of drinking, the actions of these genes, and their interactions with specific environmental risk factors should dramatically enhance our understanding of the development of alcohol use and abuse.Both genetic and environmental factors contribute to the development of patterns of alcohol use during adolescence. The relative importance of genetic and environmental factors, however, varies for different phases of alcohol use and different stages of development. Environmental influences originating both within the family and in the child’s school and neighborhood greatly influence the initiation of alcohol use. Once drinking has been initiated, however, genetic influences assume increasing importance across development, as individual differences in patterns of drinking emerge. The degree to which genetic effects contribute to adolescent drinking patterns also is subject to moderation by the environment, with factors such as religiosity, marital status, and regional residency all contributing to the relative importance of genetic and environmental effects on alcohol use.Other recent efforts have been directed toward identifying specific genes contributing to variation in drinking patterns. As scientists begin to understand more about the nature of gene–environment interactions, molecular genetic studies can be designed that take this emerging information into account . The potOnce researchers have identified specific genes associated with complex disorders such as alcoholism, they can begin to address questions regarding developmental effects or gene–environment interactions associated with these genes b. Such q


Results show that different dissociation forms of NOR had different maximum electronic absorbance wavelengths (NOR2+ < NOR0 < NOR+) and showed different photolysis reactivity. Analysis of transition states (TS) and reaction activation energies (Ea) indicated NOR+ generally underwent loss of the piperazine ring (C10–N13 bond cleavage) and damage to piperazine ring (N13–C14 bond cleavage). For NOR2+, the main direct photolysis pathways were de-ethylation (N7–C8 bond cleavage) and decarboxylation (C2–C5 bond cleavage). Furthermore, the presence of Mg2+ changed the order of the wavelength at maximum electronic absorbance (NOR+-Mg2+ < NOR0-Mg2+ < NOR2+-Mg2+) and increased the intensities of absorbance peaks of all three dissociation species of NOR, implying that Mg2+ played an important role in the direct photolysis of NOR0, NOR+, and NOR2+. The calculated TS results indicated that the presence of Mg2+ increased Ea for most direct photolysis pathways of NOR, while it decreased Ea for some direct photolysis pathways such as the loss of the piperazine ring and the damage of the piperazine ring of NOR0 and the defluorination of NOR+.The study of pollution due to combined antibiotics and metals is urgently needed. Photochemical processes are an important transformation pathway for antibiotics in the environment. The mechanisms underlying the effects of metal-ion complexation on the aquatic photochemical transformation of antibiotics in different dissociation forms are crucial problems in science, and beg solutions. Herein, we investigated the mechanisms of direct photolysis of norfloxacin (NOR) in different dissociation forms in water and metal ion Mg Antibiotics are widely used in aquaculture, animal husbandry, and medical treatment, due to the fact that they can either kill or inhibit the growth of bacteria ,2. Antibn), resulting in the aqueous presence of NOR in various dissociation states. Previous studies have indicated that the photolysis rate of NOR is greatly influenced by the pH of the solution [Photolysis is expected to play an important role in determining the fate and behavior of fluoroquinolone antibiotics including NOR in some sunlit surface waters ,16,17,18solution ,11. Howe2+ can have an effect on the photochemical behavior of organic pollutants [2+ or Mg2+. Martínez et al. [2+ occurs at a high concentration in natural water environment [2+. Thus, the mechanisms of Mg2+’s effect on the photolysis of different dissociation species of NOR urgently need to be investigated.Recently, issues related to the study of pollution resulting from the combination of antibiotics and metals have been drawing a lot of attention ,22,23. Pllutants ,24. For ironment . Followi2+ effects based on density functional theory (DFT).Obtaining experimental data can be laborious, costly, and time-consuming. Quantum chemistry calculations have been found to be efficient alternatives for predicting the environmental behavior and fate of organic pollutants, and for providing an important information on reaction intermediates or reactive species involved in chemical reactions that are difficult to be detected experimentally ,29,30,312+ in water were calculated using time-dependent density functional theory (TDDFT) at B3LYP/6-311++G  level [NOR was selected as a model compound . The geop) level ,36,37.2+ in solvent water were calculated employing the DFT method at the B3LYP/6-311+G  level of theory. The geometries at the lowest triplet states were calculated with a spin multiplicity of 3. Frequency calculations were performed at the same level to confirm all the stationary points. Transition states (TS) were characterized with one imaginary vibrational frequency. Intrinsic reaction coordinate (IRC) calculations were performed to confirm that TS do connect with the corresponding reactants and products [The possible direct photolysis reaction pathways were calculated at the lowest excited triplet states, as the lowest excited triplet states have been found to be long-lived photochemical reaction precursors for many compounds . The dirproducts . Zero-poproducts .Ka,1 (3.11 ± 0.30), pKa,2 (6.10 ± 0.19), pKa,3 (8.60 ± 0.10), and pKa,4 (10.56 ± 0.30) in water [0, NOR+, NOR2+ are dominant in natural water environments, and were thus investigated in the present study  is longer than that of NOR0 (1.486 Å)/NOR+ (1.489 Å). The bond length of C10–N13 of NOR+ (1.481 Å)/NOR2+ (1.478 Å) is longer than that of NOR0 (1.407 Å). The bond length of N13–C14 of NOR+ (1.523 Å)/NOR2+ (1.529 Å) is longer than that of NOR0 (1.471 Å). The dihedral angle O3–C2–C5–C6 of NOR+ (47.0°) is similar to that of NOR0 (52.8°). However, the dihedral angle O3–C2–C5–C6 of NOR2+ is 89.4°, indicating that the plane of O3–C2 bond is nearly orthogonal to the plane of C5–C6 bond. Additionally, the computed electronic absorption spectra of the three dissociation forms of NOR in water are shown in 2+ (280 nm) < NOR0 (282 nm) < NOR+ (308 nm) at TDDFT/B3LYP/6-311++G  level. The corresponding experimental data is around 275 nm in water (pH = 7.0) [NOR may exhibit five dissociation species with pin water , of whicnt study . The optH = 7.0)  and 274  NOR+ 1.5 Å/NOR2+ Ea, kcal·mol−1) for the five photolysis reaction pathways in different dissociation forms are listed in As depicted in Ea values of pathway R3  and R4  for NOR+ are obviously lower than those of other pathways. Thus, pathways R3 and R4 are easier to carry out than other pathways, and are the main pathways for the direct photolysis of NOR+. Additionally, the Ea value of pathway R1  for NOR+ is the highest of all the five photolysis pathways, indicating that the cleavage of the N7–C8 bond (de-ethylation) is the hardest to overcome among the five pathways.As can be seen from 2+, the Ea values of pathway R1  and R2  are remarkably lower than those of other pathways   is the lowest, while the Ea value of pathway R3  is the highest among all the pathways. Thus, NOR0 is difficult to take off the piperazine ring via cleavage of C10–N13 bond. Taken together, NOR2+ is the easiest to de-ethylate via the cleavage of the N7–C8 bond among the three dissociation species, and are easier to undergo the cleavage of the C2–C5 bond (decarboxylation) than the other two dissociation species. In addition, NOR+ is the easiest with which to carry out the cleavage of the C10–N13/N13–C14 bond among the three dissociation species. Thus, the photochemical reactivities of the different dissociation species of NOR are quite different from each other.For dissociation form NORpathways , revealil·mol−1) , indicat2+ are shown in +-Mg2+ has four possible geometries. NOR+-Mg2+ (1) is the most stable geometry with the lowest single point energy among the four geometries, and is the main geometry discussed in this research (0-Mg2+ (1) and NOR2+-Mg2+ (1) are the most stable geometries (0-Mg2+ (1), NOR+-Mg2+ (1), and NOR2+-Mg2+ (1).Optimized geometries of the complex NOR-Mgresearch . The resometries . All the+ and NOR0 is 47.0° and 52.8°  level  < NOR0-Mg2+ (266 nm) < NOR2+-Mg2+ (280 nm). Additionally, with the presence of Mg2+, there is an increase of 49.9%, 167.3%, and 108.0% in the intensities of absorption peaks for the three dissociation species NOR0, NOR+, and NOR2+, respectively. The experimental results of Martínez et al. [2+ is present. Thus, the metal ion Mg2+ has an impact on both the structures and UV absorbance spectra of different dissociation species of NOR, which may also have an impact on direct photolysis of different dissociation species of NOR.The UV absorbance spectra of complexes with Mgp) level b. The caz et al.  also sho2+ at the excited triplet states, including de-ethylation (N7–C8 bond cleavage/R1), decarboxylation (C2–C5 bond cleavage/R2), loss of piperazine ring (C10–N13 bond cleavage/R3), damage of piperazine ring (N13–C14 bond cleavage/R4), and defluorination (C11–F12 bond cleavage/R5). The computed activation energies  for the five photolysis reaction pathways in different dissociation forms are listed in Five possible reaction pathways were considered in the calculation of direct photolysis of complexes NOR-Mg+-Mg2+, the computed Ea values of four pathways  are all higher than those of monomer NOR+, indicating that Mg2+ hinders the cleavage of the N7–C8 bond, C2–C5 bond, C10–N13 bond, and N13–C14 bond  is the lowest among all the five pathways.For the complex NORC14 bond . However2+-Mg2+, Mg2+ has little impact on the Ea value of pathway R1, while Mg2+ significantly increases the Ea value of pathways R2, R3, R4, and R5  is the most easily occurring among the five direct photolysis pathways for NOR2+.For the complex NOR, and R5 . Further0-Mg2+, the Ea values of pathways R1and R2 are higher than those for the monomer NOR0, while the Ea values of pathways R3 and R4 are obviously lower than those for monomer NOR0. Mg2+ inhibits the cleavage of the N7–C8 bond and C2–C5 bond, but promotes the cleavage of the C10–N13 bond and N13–C14 bond. Furthermore, for the monomer NOR0, the cleavage of the N7–C8 bond (pathway R1) is the most easily occurring among the four photolysis pathways, but for the complex NOR0-Mg2+, the cleavage of the N13–C14 bond (pathway R4) is the most easily occurring.For the complex NOR0, NOR+, and NOR2+. As can be seen from Ea is NOR2+ < NOR0 < NOR+, which is not changed by the presence of Mg2+. For decarboxylation (C2–C5 bond cleavage/R2), the order of Ea is NOR2+ < NOR+ < NOR0, which is not changed by the presence of Mg2+. For both the loss of the piperazine ring (C10–N13 bond cleavage/R3) and the damage of the piperazine ring (N13–C14 bond cleavage/R4), the order of Ea is NOR+ < NOR2+ < NOR0, which is changed by the presence of Mg2+ (NOR+-Mg2+ < NOR0-Mg2+ < NOR2+-Mg2+). For defluorination (C11–F12 bond cleavage/R5), the order of Ea is NOR+ < NOR2+, which is not changed by the presence of Mg2+.With the increase of pH, the dominant dissociation species is, in turn, NOREa indicated that NOR0, NOR+, and NOR2+ showed disparate photochemical reactivity in water. The main photolysis pathways for NOR+ were the loss of the piperazine ring via the cleavage of the C10–N13 bond and the damage of the piperazine ring via the cleavage of the N13–C14 bond. The main pathways for NOR2+ were de-ethylation via the cleavage of the N7–C8 bond and decarboxylation via the cleavage of the C2–C5 bond. In addition, the presence of Mg2+ altered the electronic absorption spectra of NOR and increased the intensities of absorbance peaks of NOR. The TS results demonstrated that Mg2+ had dual effects, as it can inhibit or promote the direct photolysis reactions of NOR. Quantum mechanics and density functional theory are potential tools for elucidating the mechanism of photochemical transformation of antibiotics in the water environment.In summary, the computed TS and