Klebsiella oxytoca Z28 with ferulic esterase activity was screened from Daqu. In addition, the FAE gene was expressed in Escherichia coli BL21 (DE3). The enzyme consists of 340 amino acids with a molecular mass of 37.7 kDa. The FAE enzyme activity was 463 U/L when the substrate was ethyl 4-hydroxy-3-methoxycinnamate and the optimum temperature and pH were 50 °C and 8.0, respectively. The enzyme had good stability at temperatures of 25–40 °C and a pH of 8.0. Ba2+, Cu2+, Mn2+, and Ca2+ had a strong inhibitory effect on the enzyme activity, and Na+ had a promotive effect on the enzyme activity. The de-starching wheat bran was degraded by KoFAE, and the FA release was up to 227.15 µg/g. This indicated that the heterologous expression of KoFAE from Klebsiella oxytoca Z28 in E. coli had a certain potential of biodegradation, which can be applied to the degradation of agricultural waste to obtain high value-added FA products.Feruloyl esterase  can degrade biomass to release ferulic acid (FA), which has a high application in bioprocessing, food, pharmaceutical, paper, feed, and other industrial fields. A strain of  Feruloyl esterase , also known as cinnamic acid esterase, is a subclass of carboxylate esterase that hydrolyzes the ester bond break between ferulic acid  and arabinoxylan or pectin, releasing FA monomer or dimer ,3. FAE iAspergillus niger [Aspergillus oryzae [Aspergillus terreus [Lactobacillus fermentum [Lactobacillus crispatus [Bacillus pumilus [Clostridium thermocellum [Aspergillus niger has been studied more intensively, and the FAE enzymatic activity of different sources varies greatly. Although the fungal FAE enzyme activity is high, its poor stability makes it difficult to meet large-scale production applications [Butteriviribrio proteolaticus [Bacillus pumilus [Lactobacillus [Given the great potential of FAE for industrial applications, many FAE-producing microorganisms have been identified. Currently, FAE is mainly isolated from fungi such as us niger , Aspergis oryzae , Aspergi terreus , and othermentum , Lactobarispatus , Bacillu pumilus , and Clomocellum . The FAEications . Relativolaticus , Bacillu pumilus ), and Labacillus . Bacteribacillus . TherefoKlebsiella oxytoca Z28. There have been few reports of Klebsiella oxytoca FAE. Through sequence analysis, the potential FAE gene in the genome of Klebsiella oxytoca bacteria was discovered, and the expression of the gene in E.coli was realized. This enzyme has good FAE activity under alkaline conditions, and the enzyme was used to degrade starch-free wheat bran to release FA in order to lay a foundation for the combined release of FAE and lignocellulase to achieve factory production.Bacteria FAE produced by bacteria has been reported to have good FAE under acidic conditions. However, less has been reported about alkaline FAE. In this study, the Z28 strain with the highest FAE enzyme activity was screened from 37 strains in Daqu wine and distiller’s grains that had a degrading effect on ethyl 4-hydroxy-3-methoxycinnamate and identified as 4)2SO4, KH2PO4, MgSO4·7H2O, CaCl2·H2O, and FeCl3 were purchased from Kelong Chemical Co., Ltd. ; ferulic acid and ethyl 4-hydroxy-3-methoxycinnamate were purchased from Yuanye Biotechnology Co. Ltd. .Daqu liquor and distiller’s grain were collected from a distillery in Yibin, China; peptone, yeast extract, NaCl, : NaCl 10, yeast extract 5, peptone 10, and 5 mL ethyl 4-hydroxy-3-methoxycinnamate solution, pH 6.5. The composition of the screening medium used to assay FAE enzyme activity was (g/L): ammonium sulfate 1.3, KH2PO4 0.37, MgSO4-7H2O 0.25, CaCl2-H2O 0.07, FeCl3 0.02, yeast extract 5.0, and 5 mL ethyl 4-hydroxy-3-methoxycinnamate, pH 6.5. All media were sterilized at 121 °C for 20 min.The medium used for enrichment of microorganisms was an LB medium; the composition of the medium used for preliminary screening of microorganisms was (g/L): NaC1 0.3, ammonium sulfate 1.3, MgSOv/v) seed solution was inoculated by an in vitro enzyme-producing medium and cultured in a shaker at 37 °C and 180 rpm for 48 h. A total of 200 µL of the fermentation broth was placed in the Oxford cup added to the initial screening medium, and the medium was incubated at 37 °C for 48 h. The ability of FAE in the fermentation broth to decompose ethyl 4-hydroxy-3-methoxycinnamate was observed. The capacity of FAE production and FAE activity were preliminarily determined by the size of the hyaline ring.A total of 10 g of Daqu and distiller’s grain were weighed and added to a 250 mL conical flask. Each of them was added to a 250 mL conical flask with 90 mL of physiological saline and shaken in the conical flask for 30 min. The suspension was diluted to a certain number of times and then applied to the medium containing ethyl 4-hydroxy-3-methoxycinnamate and incubated in a constant temperature incubator at 37 °C for 72 h to observe the degradation of ethyl 4-hydroxy-3-methoxycinnamate by the strain. The strains with a larger circle size were selected for isolation and purification 3 times and then stored at −80 °C for storage. The seed solution was obtained by inoculating the preserved strain in the LB liquid medium for activation. The preserved strains were inoculated in the LB liquid medium and cultured in a shaker at 37 °C and 180 rpm for 24 h to obtain seed solution. The 2%  buffer solution containing 1% ethyl 4-hydroxy-3-methoxycinnamate. After allowing the enzyme to react with the substrate for 2 h at 37 °C, the reaction was terminated in an ice bath for 10 min. Ferulic acid production was measured at 320 nm by ultraviolet spectrophotometer. The above methods were compared with substrates without enzymes.FAE-producing strains were inoculated in the LB medium for 12 h after incubation at 37 °C, and colony morphology and a Gram stain were observed. The genome of the strain was extracted using the TIANamp Bacterial DNA Genome Extraction Kit. PCR amplification of the 16S rRNA gene was then performed, using universal primers of 27-F (5′-CAGAGTTTGATCCTGGCT-3′) and 1492-R (5′-AGGAGGTGATCCAGCCGCCGCA-3′). The PCR products were detected by 1% agarose gel electrophoresis and sent to Chengdu Qingke Company for sequencing. Sequencing results were compared with microorganisms in the GenBank database using the Blast created by the NCBI. Highly similar strain sequences were selected, and a phylogenetic tree was constructed using the neighbor-joining method (500 bootstrap copies) of MEGA 7 software.Klebsiella oxytoca Z28 genomic DNA as a template, and the upstream and downstream primers . The PCR products were purified to obtain more pure DNA fragments. The purified DNA fragment was digested with BamHI and XhoI, and then the digested DNA fragment was connected to pET-28a to construct the recombinant plasmid pET-28a-KoFae. It was transformed into E.coli DH5α, and the plasmid was extracted and identified by double enzyme digestion. Plasmids identified as positive were sent to Qingke Biological Company for sequencing. The sequencing results of the sequence can be seen in the E. coli BL21 (DE3), and the recombinant E.coli BL21/pET-28a-KoFae was cultured overnight and activated. The cultured seed solution was inoculated with LB medium containing resistance (50 μg/mL kanamycin) at 2% volume. It was incubated at 37 °C, 180 r/min for 2–3 h and then added 1 mM isopropyl beta-d-thiogalactoside (IPTG). It was then incubated at 25 °C, 180 r/min, for 8 h. Recombinant E. coli BL21/pET-28a-KoFae was collected by centrifugation at 4 °C and 7000 rpm. The cells were washed twice with 20 mm PBS (pH 7.4), resuspended, and ultrasonically broken. After centrifugation at 4 °C and 7000 rpm for 30 min, the supernatant was collected as a KoFAE enzyme crude solution.The KoFae gene (Genbank accession number: WP_105889557) was amplified by PCR using E. coli containing pET-28a for the same time was used as a blank control. The residual enzyme activity was detected after the crude enzyme solution was kept at different temperatures for 1 h. The initial enzyme activity of the enzyme at different temperatures was 100% to determine the effect of temperature on the stability of the enzyme. Citrate–sodium citrate buffer (pH 3.0–5.0), phosphate buffer (pH 6.0–8.0), and glycine–sodium hydroxide buffer (pH 9.0–10.0) were prepared. The activity of FAE was measured under different pH conditions to determine the optimum pH of the enzyme. The residual enzyme activity of the crude enzyme solution was detected after it was stored at different pH for 1 h. The initial enzyme activity at different pH values was 100% to determine the effect of pH on enzyme stability.The crude enzyme solution of KoFAE was reacted with the substrate ethyl 4-hydroxy-3-methoxycinnamate at the temperature of 25–60 °C to determine the optimum reaction temperature of the enzyme. The crude enzyme solution induced by 2+(CoCl2), Ba2+(BaCl2), Na+(NaCl), Cu2+(CuSO4), Zn2+(ZnSO4), Fe3+(FeSO4), Li+(LiCl), Mn2+(MnCl2), Mg2+(MgSO4), and Ca2+(CaCl2)) buffer solution. The crude enzyme solution induced by E. coli containing pET-28a for the same time was used as a blank control. After storage at 4 °C for 30 min, the enzyme activity was measured to determine the effects of metal ions on the enzyme activity.The obtained crude enzyme solution was mixed with a final concentration of 1 mM metal ions (Cow/v) amylase solution at 65 °C for 1 h with frequent stirring. Then, the solution was boiled at 99 °C for 30 min to inactivate the enzyme. Finally, the bran was repeatedly washed with distilled water to completely remove the starch. The obtained de-starch bran was dried to a constant weight. FA was produced from de-starching wheat bran by KoFAE. A total of 0.5 g wheat bran was suspended in 30 mL of 100 mM phosphate hydrochloric acid buffer (pH 8.0). In total, 10 mL KoFAE enzyme solution was added to the reaction system and incubated for 10 h at 150 r/min at 50 °C. After incubation, the reaction was terminated by boiling for 30 min and centrifuged at 8000× g for 10 min. The supernatant was collected and filtered for LC-MS analysis. LC-MS conditions: Waters Acquity UPLC Beh C18 1.7 μm 2.1 × 50 mm; eluent: 10% 0.1% acetic acid water, 90% methanol, column temperature 25 °C, flow rate 0.2 mL/min; mass spectrometry conditions: TEM 500 °C, IS-4500v, CUR 25psi, GS1 50 psi, GS2 50 psi, detection mode MRM, FA 192.9 > 134, DP-43v, EP-11V, CE-16V, CXP-5V.Deamylated bran was prepared as follows: 100 g of bran was mixed with an appropriate amount of water, boiled in a boiling water bath for 20 min to melt the starch, rinsed repeatedly with distilled water, and incubated in a 0.5%  has been submitted to the GenBank nucleotide sequence database (registration number: WP_105889557). The KoFae gene was amplified by PCR using the Klebsiella oxytoca Z28 genome as the template, and the characteristic band appeared at about 1023 bp. The PCR product was ligated onto the pET-28a vector, and the target band of about 1023 bp was identified by double enzyme digestion. The sequencing results were consistent with the results published by GenBank. The KoFae open reading frame was 1023 bp, encoding 341 amino acid residues. The molecular mass and pI were 36,127.33 kDa and 7.81, respectively . The protein is named KoFAE. The grand average of the hydropathicity of koFAE was −0.049, indicative of a hydrophilic protein. By comparing KoFAE with FAE protein sequences previously reported, the results showed that the homology of Kofae with FAE derived from Klebsiella pneumoniae (GenBank ID: BCI97608.1) was the highest 40.74%. The homology between KoFAE and FAE with Lactobacillus crispatus (GenBank ID: MK640209.1) and B. altitudinis (GenBank ID: AKC64980) was 39.58% and 31.31%, respectively. In addition, it was found that a “Gly-X-Ser-X-Gly” motif was observed in the sequence, which was highly conserved in previously reported FAE. E.coli BL21(DE3) was transformed with the pET-28a-koFae plasmid, and after 0.5 mmol/L IPTG induced expression, SDS-PAGE electrophoresis detected the target band at about 36.13 kDa (E.coli BL21(DE3) containing pET-28a and pET-28a-KoFae were inoculated onto plates containing ethyl 4-hydroxy-3-methoxycinnamate  [Penicillium piceum (70 °C) [Lactarius hatsudake (30 °C) [Lactobacillus has good thermal stability at the temperature of 25~50 °C. KoFAE in this study had good thermal stability when the temperature ranged from 25 °C to 40 °C. When the temperature increased from 45 °C to 50 °C, the stability decreased slowly, and more than 60% of the enzyme activity still remained. The optimal reaction pH for KoFAE is shown in B. intestinalis. It has good activity and stability at pH 5.5, but the FAE enzyme activity is less than 10% at pH 8.0 [The optimal temperature and temperature stability of KoFAE were evaluated A,B. When (37 °C) , Penicil (70 °C) , and Lac (30 °C) . Xu et a (30 °C)  found th2+, Cu2+, Mn2+, and Ca+ inhibited the activity of the Kofae enzyme by more than 40%. However, Co+, Zn2+, Fe3+, and Mg2+ inhibited the enzyme activity by about 20%, while Na+ had a slight promotion effect on the enzyme activity. Different from previous studies, Agrawal et al. found that Mg2+, Ca+, and Cu2+ promoted FAE derived from Pichia pastoris X33, while Na+ inhibited FAE [The effects of metal ions on the enzymatic activity of KoFAE were measured . The resited FAE . This reLactobacillus-derived FAE to release up to 199 μg FA from 0.2 g de-amylated wheat bran. In this study, FA can be obtained in a short time by using FAE derived from Klebsiella oxytoca, which promotes the development of the research on the release of FA from the degradation of lignocellulosic waste. According to the characteristics of this enzyme, which belongs to alkaline FAE, subsequent studies can study its ability to release FAE from waste under alkaline conditions, so as to meet the actual industrial demand.An important current application of FAE is the degradation of lignocellulose to release FA . De-starKlebsiella oxytoca Z28 with FAE activity was obtained from the liquor Daqu, and its FAE activity reached 35.0 U/L. In addition, for the first time, an alkaline FAE was identified from Klebsiella oxytoca and heterologous expressed in Escherichia coli. The enzyme activity of the crude KoFAE enzyme solution reached 463 U/L when ethyl 4-hydroxy-3-methoxycinnamate was used as substrate. The enzyme was stable at 25~40 °C and pH 8.0, and Na+ could activate the activity of KoFAE. Using the crude KoFAE enzyme solution directly on de-amylated wheat bran, the FA release could reach 227.15 µg/g. This work enriched the resources of ferulic acid-releasing strains, provided an FAE derived from Klebsiella oxytoca, and provided an experimental basis for bioextraction of FA from agricultural waste species.In this study, a strain of


When “hijacked” by compulsive behaviors that affect the reward and stress centers of the brain, functional changes in the dopamine circuitry occur as the consequence of pathological brain adaptation. As a brain correlate of mental health, dopamine has a central functional role in behavioral regulation from healthy reward-seeking to pathological adaptation to stress in response to adversity. This narrative review offers a spotlight view of the transition from healthy reward function, under the control of dopamine, to the progressive deregulation of this function in interactions with other brain centers and circuits, producing what may be called an anti-reward brain state. How such deregulation is linked to specific health-relevant behaviors is then explained and linked to pandemic-related adversities and the stresses they engendered. The long lockdown periods where people in social isolation had to rely on drink, food, and digital rewards via the internet may be seen as the major triggers of changes in motivation and reward-seeking behavior worldwide. The pathological adaptation of dopamine-mediated reward circuitry in the brain is discussed. It is argued that, when pushed by fate and circumstance into a physiological brain state of anti-reward, human behavior changes and mental health is affected, depending on individual vulnerabilities. A unified conceptual account that places dopamine function at the centre of the current global mental health context is proposed. The neurotransmitter dopamine fulfills a critical function in regulating the responses of the mesolimbic system, also known as the reward system, in the neural circuits of the mammalian brain . The rewA reward requires the coordinated release of heterogenous neurotransmitters, where dopamine plays the central role by mediating the reward value of food, drink, sex, social interaction, or specific substances or stimuli, as in abuse and addiction ,7. MoreoA reward is what we are all after every day, since the dawn of time, governed by a natural process where the brain associates so-called stimuli  with a positive, desirable, outcome ,13,14. AThe release of dopamine can likewise be produced by “unnatural rewards” such as alcohol, cocaine, methamphetamine, heroin, nicotine, marijuana, and other substances or by compulsive activities such as gambling, eating, sex, and risk-taking behaviors . Drugs canhedonia [anhedonia through its interaction with the glucocorticoid system, responding to chronic stress. Prolonged stress leads to inflammatory processes that negatively impact functional connectivity in the corticostriatal (dopaminergic) reward circuits and symptoms in close interaction with the endocannabinoid and orexin systems [Anhedonia, i.e., the inability to experience pleasure, is thus a direct consequence of our brain’s response to chronic stress [As an addictive behavior becomes more and more of a habit that cannot be controlled by willpower, the reward system becomes increasingly networked with other brain areas that control different aspects of habitual behavior ,24. Thisnhedonia ,37. The nhedonia , where e systems . Throughc stress ,39,40 anc stress  have shoc stress . Allostac stress ,44. Inflc stress ,46. IncrThe important role of dopamine in the neurocircuitry of the reward system and its interactions withotherbrain systems provide a functional (brain-based) interpretation of the changes in health-relatedbehavior in light ofthe conceptual framework of the neurobiology of motivation. The resources of modern neuroscience offer an opportunity to conceptualize the dopaminergic neurocircuitry and its multiple interactions with other pathways, functions, and mechanisms in healthy rewards; in addictive processes; and in the development of chronic stress, anhedonia, and ultimately, depression. Changes in motivation that are associated can be generalizedforother psychopathological processes. Therein, compulsory reward-seeking and, ultimately, addiction may be conceptualized as a cycle of decreased function of the brain’s reward system and the recruitment of an anti-reward system. As a consequence, the normally homeostatic function of reward fails, as compulsivereward-seekingin the shortterm results in the amelioration of a sudden reward deficit but, ultimately, in the long term, produces neurochemical dysregulations ofdecreased dopamine and opioid peptide function, increased corticotropin-releasing factor, and a chronic deviation of the reward system that is fueled not only by dysregulation of the reward circuits but also by the recruitment of other neurotransmitter and hormonal stress responses ,47. OnceCompulsive behavior consists of repetitive acts driven by the urge that one has to perform them while aware that they are not in line with one’s overall goal . There iA craving is a particual aspect of compulsive behaviour, andbingeing is a direct result of craving. Preoccupation, a motivation-related mental state triggered by aversive internal or external stimuli, may produce compulsive reward-seeking in healthy and in addictive behaviours ,69,70 thMotivational changes related to the sudden withdrawal of a reward ,59 in boallostasis) that lead to irritability, dysphoria, anxiety, and anhedonia in what is sometimes called the brain’s anti-reward system [While healthy, positive stimulation of the reward system promotes healthy sleep, compulsive reward-seeking, on the other hand, leads to the asynchronization of the circadian rhythm ,81,82,83d system ,85,86. Bd system ,17. A nad system ,32. Dopad system ,14,17,31d system  resultind system . Most imd system .➢significant rise in addictions and related mental illnesses;➢significant rise in the corresponding anti-depressant prescription uptake;➢increased risk of suicidal ideation or suicide.In 2022, the World Health Organisation (WHO) issued a brief  with facanhedonia linked to compulsive or addictive behaviors such as binge-eating [Any potential doubts about the impact of COVID-19 on mental health have been largely put to rest by the most recent papers on the topic ,92,93,94e-eating ,96, subse-eating , and dige-eating  were idee-eating ,99,100—iMental health issues arise from a specific context. Recently reported effects of the pandemic have shown, beyond all reasonable doubt, that adversity affects people’s capacity tocope with the stresses it produces. Conditions of extreme adversity challenge the stress and immune system responses needed for coping, and beyond a certain threshold, such conditions can trigger behavioral changes as listed and discussed here above; all of them have been functionally linked to the complex brain pathways involving dopamine release, reward mechanisms, and their interactions or regulations ,79. DuriThe COVID-19 pandemic creatednegative health consequences such as consuming high-fat and sugar foods, increasing body weight , anxietyCOVID-19 spread across the world ata rapid pace, and to further limit the spread of infection, lockdowns were declared in most parts of the world. People were forced to stay indoors, and the internet was the only source of entertainment. This context engendered addictive behavior with measurable negative effects on anxiety and sleep quality, especially among younger individuals ,117,118.Dopamine Nation [In her book e Nation , the psyFuture research on dopamine interactions, from the molecular, cellular, and integrative brain circuitry levels, and their underlying behaviors has acquired a novel global significance in this new mental health context ,143,146.


In Response: Real-time evaluation of predictive models for antimicrobial resistance (AMR) is critical for real-world applications, as indicated in our recently published article  that should be included in the model? Second, how will healthcare providers and hospitals use AMR forecasts? What policies will be informed by forecasts, and what are the downstream effects? Answers to those questions will help determine the eventual real-world utility of predictive models.Evaluating real-time AMR prediction is a complicated task. By drawing experience from computer vision (


Antimicrobial resistance (AMR) is a global public health threat, especially in low-resource settings. Irrational and inappropriate use of antibiotics in healthcare settings has led to widespread drug resistance. Antibiotics are widely and inappropriately used without adherence to international guidelines in Bangladesh, which may contribute significantly to the development of antimicrobial resistance (AMR). With the rapid raising of AMR, it is important to assess antibiotic prescribing perceptions and practices among physicians in Bangladesh.From September to December 2020, a hospital-based cross-sectional survey was conducted among 517 physicians in 11 tertiary care hospitals across Bangladesh. Face-to-face interviews were done to collect information. Descriptive statistics were performed to analyze the data and for interpretationand antibiotic cost. All (100%) physicians stated that updated antibiotic use guidelines are necessary for the appropriate and rational use of antibioticsNearly two-thirds (66 %) of the physicians considered AMR a significant concern. Over three-fourths (77%) opined that easy access to antibiotics without a prescription was the most important contributor to AMR, followed by overuse/inappropriate use (68%); whereas 42% of physicians reported inadequate diagnostic tests and one-fifth blamed poor infection control measures as the cause of rising AMR. The majority (77 %) of the participants reported following senior physicians’ guidance to prescribe antibiotics while 41.6% prescribe considering the affordability of patients The assessment's findings can aid policymakers in the development and implementation of a national guideline for ensuring the rational use of antibiotics and enforcement of drug regulations at all stages, including the dispense of antibiotics in Bangladesh.All Authors: No reported disclosures


These results were confirmed significant even after adjustment for GA and for the ongoing modality of respiratory support.This prospective observational study aimed to evaluate whether lung fluids, assessed by lung ultrasonography and transthoracic electrical bioimpedance (TEB), may be influenced by the presence of a haemodynamically significant patent ductus arteriosus (hsPDA) in very preterm infants during the transitional period. Infants < 32 weeks of gestational age (GA) admitted to the neonatal intensive care units of IRCCS AOU Bologna and Niguarda Metropolitan Hospital of Milan  underwent a daily assessment of a lung ultrasound score (LUS) and of a TEB-derived index of thoracic fluid contents (TFC) during the first 72 h after birth. Echocardiographic scans were simultaneously performed to evaluate the concomitant ductal status (hsPDA vs. restrictive or closed duct). The correlation between LUS, TFC, and the ductal status was tested using generalized estimating equations. Forty-six infants  were included. At each daily evaluation, the presence of a hsPDA was associated with significantly higher LUS and TFC compared with a restrictive or closed ductus ( Conclusion: Even during the first 72 h of life, the presence of a hsPDA determines a significant increase in pulmonary fluids which can be non-invasively detected and monitored over time using lung ultrasonography and TEB. The reabsorption of lung fluids is crucial for the respiratory adaptation to extrauterine life. Together with the primary surfactant deficit, fluid retention in the alveoli ensuing from the immaturity of the related clearance systems is a major contributor to the development of respiratory distress syndrome (RDS) in preterm neonates.The presence of a haemodynamically significant patent ductus arteriosus (hsPDA) with an excess left-to-right transductal shunting is frequent among very preterm infants during early postnatal phases. Consistently with the resulting pulmonary overflow, which contributes to the accumulation of alveolar fluids, hsPDA has been associated with a fourfold increase of RDS risk .Monitoring lung fluid clearance in preterm infants with RDS and hsPDA may provide potentially useful information for their therapeutic management. Lung ultrasonography is widely used for non-invasive assessment of lung fluids in preterm neonates . RecentlThis study aimed to evaluate whether the presence of a hsPDA in very preterm infants during the transitional period may influence lung fluids, assessed non-invasively by lung ultrasonography and TEB.The present study involves a sub-analysis of a prospective observational research conducted at the neonatal intensive care units (NICUs) of IRCCS AOU Bologna  and Grande Ospedale Metropolitano Niguarda  from May 2020 to January 2022. Infants < 32 weeks’ gestational age (GA) with RDS, defined by an acute respiratory failure with onset within the first 24 h of life, accompanied by evidence of the presence of diffuse, bilateral, irregular lung opacities or infiltrates at chest radiography (if performed) and responsive to lung-recruiting ventilatory strategies and/or to surfactant treatment , were inDuring the first 72 h of life, the enrolled infants underwent daily assessments of lung ultrasonography and echocardiography. Lung ultrasound findings were classified using a validated lung ultrasound score (LUS) assigning the following scores to six pulmonary fields: 0: only A-lines or < 3 B-lines; 1: ≥ 3 B-lines; 2: crowded and confluent B-lines with or without consolidations; 3: significant consolidations . Echocar®, Osypka Medical GmbH, Berlin, Germany) with a beat-to-beat sampling frequency. After the recording, the traces were checked to rule out potential artifacts; the signal goodness was also assessed to improve artifact detection as previously described ), and between-group comparisons of daily LUS, TFC, and LVO values were performed using the Mann–Whitney U test. To account for repeated measurements on each subject, the independent effect of hsPDA on TFC and LUS, net of such influencing covariates as GA and the modality of respiratory support, was evaluated using generalized estimating equations (GEEs). Since the ductal features and the modality of respiratory support changed over the study period, these variables were handled as time-dependent covariates, and their daily status was included in the GEEs. Statistical analysis was performed using SPSS version 27 . Significance level was set at 2O. All the infants requiring invasive ventilation received endotracheal surfactant before the first LUS and TFC assessment; for this reason, this variable was not included in the multivariable analysis.Forty-six preterm infants  were included. All the included infants were longitudinally sampled on days 1, 2, and 3. The prevalence of a hsPDA on days 1, 2, and 3 was 26/46 (56.5%), 14/46 (30.4%), and 8/46 (17.4%), respectively; in 4 infants, pharmacological closure of the hsPDA was undertaken on day 3 due to the occurrence of systemic hypotension requiring inotropes. Forty-three (93.5%) out of 46 infants had echocardiographic evidence of a foramen ovale. Invasive ventilation was required by 14/46 infants (30.4%) on day 1, 12/46 (26.1%) on day 2, and 11/46 (23.9%) on day 3. Of the 14 infants who required an invasive respiratory support on day 1, 7 were placed on conventional modalities and 7 on high-frequency oscillatory ventilation with a median MAP of 11 cmHAs detailed in Table p < 0.001) and TFC  compared with a restrictive or closed ductus; estimated mean values of both parameters in relation to the ductal status are illustrated in Fig. p = 0.018) and TFC , whereas an inverse relationship between TFC and GA was observed .The GEE results confirmed that the presence of a hsPDA significantly increased LUS (β = 2.821 [95%CI 1.315–4.326], According to the present results, a combined monitoring of lung ultrasonography and TEB in very preterm neonates during the transitional period may effectively detect the effects of a hsPDA on lung fluids. Compared to a closed or restrictive ductus, the presence of a hsPDA was independently associated with significantly higher LUS and TFC values, suggestive of increased lung fluids. As reflected by the increased LVO observed in hsPDA infants, this finding is consistent with the effects of the pulmonary overflow resulting from the excess left-to-right transductal shunting.Lung ultrasonography has been largely adopted in neonatal care to assess infants with RDS , 11. TEBEvidence of lung fluids accumulation in infants with a hsPDA since the early postnatal phase may entail potentially relevant clinical implications. Since the lung fluid accumulation ensuing from the transductal shunting may contribute to the severity of respiratory distress or to its persistence, infants with RDS and echocardiographic evidence of a hsPDA may benefit from a careful balance of fluid intakes during the postnatal transition. A recent survey investigating fluid management in preterm infants with a hsPDA has highlighted a lack of standardization and wide variability among Italian NICUs ; in thisThe association between hsPDA persistence and later BPD development has been largely documented . We haveThe small study sample represents needs to be acknowledged as a main limitation. Although similar criteria for fluid management, hsPDA closure, mechanical ventilation, extubation, and surfactant administration were adopted in the two centers, the bicentric nature of the study may limit the generalizability of the present results, which, therefore, need to be validated on larger samples. Moreover, the possible impact of ventilation parameters on LUS and hsPDA could not be assessed in the present study, thus posing a potential bias.In conclusion, the presence of a hsPDA can have a significant impact on pulmonary fluids since the early postnatal phase. Lung fluid clearance in preterm infants with RDS and a hsPDA can be quantitatively monitored using such non-invasive techniques as lung ultrasounds and TEB, which may add potentially useful information for an individualized management of this population. Nevertheless, further targeted studies are required to validate these findings and to assess their pathophysiological impact on later pulmonary outcomes.


To solve the environmental disaster that is generated by legacy plastics accumulation, researchers are looking to design plastics with enhanced end-of-life options, but many circular plastics do not meet industrial requirements. Here, we highlight a metal-free approach to produce chemically recyclable poly with ultra-high molecular weight and comparable properties to one of the most produced plastics, polyethylene. CRM mitigates the need for continuous feedstock sourcing and could potentially minimize the accumulation of plastic waste. While initial reports on CRM plastics did not specifically focus on the potential scalability of these materials, in recent years, special attention has been paid to the design of easily scalable systems, accessing useful material properties, and invoking simple processes for depolymerization to monomers. Polyacetals are promising candidates for CRM and, recently, a reversible-deactivation cationic ring-opening polymerization (CROP) of cyclic acetals using a commercial halomethyl ether initiator and an indium(III) bromide catalyst was introduced7. Using this method, poly (PDXL) of around 200 kDa was produced, obtaining materials with tensile strengths comparable to some commodity polyolefins that could be chemically recycled in near-quantitative yield in the presence of strong acids. So far, sustainable catalysts/initiators that could transform CRM plastics into ultra-high molecular weight plastics with unique toughness thanks to their superior entanglement have been lacking.One alternative to transition from our current linear plastic production to a more sustainable circular plastic production is to design plastics capable of chemical recycling to monomers (CRM)8. The oxonium (Meerwein) salt initiators with non-nucleophilic counter-anions generate small concentrations of propagating cations. “We realized that transacetalization, a side-reaction during CROP of cyclic acetals, could be used as a mechanism for sharing propagating cationic species among different polymer chains,” says Abel. “This means that even if a propagating polymer chain terminates, this transacetalization ‘side-reaction’ revives the dead chain by transferring the propagating cation from a living chain to the dead chain. This key insight was crucial to developing the present cyclic acetal CROP system”. Because of the living nature of the polymerization, extremely low initiator concentrations i.e., monomer to initiator ratios of up to 40,000: 1 suffice for initiating the CROP of cyclic acetals. “We showed that the use of DTBP as a proton trap during the CROP of cyclic acetals prevents undesired initiation by acidic impurities and is crucial to achieving the molecular-weight control needed to reach ultra-high molecular weights,” says Abel. The team found that the increase in molecular weight leads to a tremendous improvement of the physical performance with maximum values for tensile stress at break of 105 MPa. The material is ductile and tough and shows a higher ultimate stress tolerance than ultra-high molecular weight polyethylene (UHMWPE) (molar mass ∼5000 kDa). “Our method of repolymerization of recycled DXL enables the synthesis of pristine PDXL with different molecular weights, mechanical properties, and architectures without affecting future recycling of the polymer,” explains Abel.Now, H. Grace Hester, Brooks A. Abel, and Geoffrey W. Coates from Cornell University, USA, employ commercially available Meerwein salts as organic initiators together with 2,6-di-tert-butylpyridine (DTBP) as a proton trap to synthesize ultra-high molecular weight PDXL of up to 2000 kDa from recycled DXL (10.1021/jacs.3c01901)Industrially viable plastics capable of CRM could bring a paradigm shift to solve issues arising from the current unsustainable plastics production system. Proper catalyst/initiator selection may well facilitate the development of such unique sustainable polymers. Furthermore, chemical diversification in the field of CRM is crucial to cover the many applications where legacy plastics are present. Other physical aspects such as molecular weight are also critical to enhance the field of plastic materials. “We are now taking steps to synthesize polyacetals of varying polymer architectures to enable further tuning of polymer properties to fit a particular application,” says Abel.


Nucleic acid amplification testing facilitates the detection of disease through specific genomic sequences and is attractive for point-of-need testing (PONT); in particular, the early detection of microorganisms can alert early response systems to protect the public and ecosystems from widespread outbreaks of biological threats, including infectious diseases. Prior to nucleic acid amplification and detection, extensive sample preparation techniques are required to free nucleic acids and extract them from the sample matrix. Sample preparation is critical to maximize the sensitivity and reliability of testing. As the enzymatic amplification reactions can be sensitive to inhibitors from the sample, as well as from chemicals used for lysis and extraction, avoiding inhibition is a significant challenge, particularly when minimising liquid handling steps is also desirable for the translation of the assay to a portable format for PONT. The reagents used in sample preparation for nucleic acid testing, covering lysis and NA extraction , are reviewed with a focus on their suitability for use in PONT. Emerging infectious diseases and their potential for worldwide outbreaks have always threatened the global public’s well-being. For example, the recent pandemic was caused by highly contagious pathogens like SARS-CoV-2, which spread exponentially at a growth rate of 0.19–0.29 per day in many countries, burdening healthcare systems and inflicting death and economic damage ,4,5,6.PONTs are designed to be performed on-site by any user, providing an accurate and rapid (minutes) screening . MicroflTo diagnose infection with a specific target , PONT assays and devices employ specific and sensitive molecular techniques, such as immunoassays (IAs) and nucleic acid amplification tests (NAATs) ,4,6. WhiAdvances in isothermal amplification techniques include loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). These innovations have successfully alleviated the engineering challenges traditionally associated with the gold standard thermocycling polymerase chain reaction (PCR). PCR relies on thermocycling between temperatures of 65 and 95 °C, requiring stringent temperature control. In contrast, isothermal amplification methods operate at a single temperature, typically between 37 and 65 °C. Detailed reviews regarding advanced amplification technologies and associated detection approaches can be found elsewhere ,11, withTypically, the sample preparation process can be divided into two stages: cell lysis and NA extraction . During Cell lysis is the first step of sample preparation. Its purpose is to release target NAs from biological samples by disrupting the structure of cell membranes, which are also known as phospholipid bilayer membranes or plasmalemma. These membranes are part of the cell’s cytoskeleton and control the transport of materials in and out of the cell, as well as communication with other cells . The upcDetergents (or surfactants) break down the phospholipid bilayer by virtue of their amphiphilic properties. The membrane solubilisation induced by detergents can be understood in three stages ,56,57. IDetergents can be ionic and non-ionic, depending on the nature of the polar head. Ionic detergents have charged polar head groups, either positively charged (cationic) or negatively charged (anionic). Cationic detergents often contain ammonium or pyridinium head groups and are used in DNA extraction and cell lysis because the positively charged nature helps disrupt cell membranes and solubilize biomolecules. While anionic detergents, commonly with sulphate or carboxylate ions, are often used in protein electrophoresis, non-ionic detergents have uncharged polar head groups and are suitable for a wide range of applications where ionic interactions should be avoided.v/v) solution with the sample diffusion owing to the laminar flow regime. Complete lysis of green fluorescence protein (GFP)-expressing cells was achieved within 1 min  ethanol) was used for cell lysis in a capillary, mixing a 0.1%  NaCl to 1.0% with the addition of 0.008% of Q. saponaria . F. FE. col samples .Advantages of chaotropic lysis include the fact that it can be performed at room temperature using a short incubation time  and that it can aid in binding the NAs to a stationary phase for NA extraction . HoweverSalmonella enterica serovar Typhimurium, from human urine and fresh milk samples, comparable outcomes to a commercial kit were obtained, reporting a LOD of 5 CFU/10 mL from both sample matrices  [Ni(hfacac)3−]) and IL  were used to lyse different plant species  within 30–60 s without an additional lysing reagent or heating. Owing to their solvating properties, the NAs were extracted into the MIL with the loaded MIL retained with the help of a magnet allowing for removal of the sample matrix and introduction of the amplification reagents. The MIL facilitated the extraction of 0.5–4 µg DNA from 0.5 mg plant tissue, more than the maximum of 0.6 µg when using  [NTf2−] [Ionic liquids (ILs) have unique solvating properties and have also been used for lysis of white blood cells and used for the extraction of NAs without significant interference with amplification. ILs are salts with a melting point < 100 °C and hence are liquid at room temperature. Magnetic ILs (MILs) are a subclass of ILs that include a paramagnetic ion . The hyd [NTf2−] . The MILE. coli) which are typically fast to lyse; hence, further testing on more sample types is desirable. ILs also allow for fast lysis and provide a greener alternative to many organic solvents, but the high viscosity . . HRAS geFor the cationic stationary phases, washing is typically conducted in PBS, at a pH maintaining the protonated nature of the stationary phase as required for DNA binding , whereasMycobacterium tuberculosis (MTB) . . 110]. CIn addition to the use immiscible liquids, gels have also been used as immiscible barriers to separate reagent conditions. An organogel-based hydrophobic purification system was presented as alternative with the potential to decrease carryover. An organogel is a gel where the fluid phase is an organic liquid, and it can be made from low-molecular-mass organogelators (LMOGs), lime 12-hydroxystearic acid (12-HAS), and an apolar liquid-like methylphenyl silicone oil, as used in The use of immiscible barriers has provided an appealing approach in translating workflows from a benchtop to a fluidic platform. Most popularly applied using immiscible interfaces between liquids, IFAST has demonstrated to be highly effective for silica beads in combination with a high concentration (3–6 M) of GuSCN or GuHCl for binding. With the rise in popularity of carboxylic acid and cationic stationary phases for the use of more benign lysis reagents as discussed in The immiscible barrier provides an effective way to remove low-molecular-weight alcohols used in washing, which is a requirement as failure to perform this can lead to inhibition of the amplification reaction ,169. An 260/A280, reaching 1.86 with a recovery rate of 10.2 ± 4.03% (sputum) and 91.2 ± 7.46% (urine). Considering operational aspects in a low-resource environment, a simplified kit for the extraction of cfDNA was presented in the form of a tube-based purification assay, employing three buffers: (1) 3 M GuSCN, 30% IPA, and 5% Tween 20; (2) 80% ethanol; and (3) 100% ethanol . From 10In the development of a PONT device, minimizing the number of reagents and processing steps is important for success. It is therefore no coincidence for many entries in PONT assays with smaller elution volumes have demonstrated comparable performance when benchmarked with conventional extraction kits. Two devices, a multiplexed centrifugal microfluidic system for SPE from buccal swabs  and an ISalmonella spp. In pork meat samples by pumping the reaction mix directly into a chamber where the dried beads with extracted DNA were located [Ni(hfacac)3−]) and IL  were used for the lysis and NA extraction from plant samples. Owing to their solvating properties, the NAs were extracted into the MIL and retained with the help of a magnet allowing for the removal of the sample matrix and introduction of the amplification reagents [In addition to the SPE-based methods, progress has also been reported on the extraction of NAs without the use of stationary phases, and an overview is provided in reagents . The NAsHydrogels are swollen polymer networks that provide size selectivity in molecular transport. This selectivity has been used to allow for the transport of NAs, while preventing the transport of inhibitors, effectively allowing for extraction of the NAs from their environment. A nanoporous crosslinked PEG hydrogel, shown in Isotachophoresis (ITP) is an electrophoretic separation and preconcentration technique that leverages a heterogeneous buffer system to generate electric field gradients for the simultaneous focusing and separation of ionic species based on their effective electrophoretic mobilities. ITP-based purification was demonstrated for extracting genomic DNA from whole blood lysate  . ITP wasITP has also been used on paper microfluidic devices. Using the negative charge to generate an electro-osmotic flow rather than for binding, a paper fluidic device was used for processing a 200 μL sample in approximately 6 min, resulting in a 20,000-fold increase in NA concentration . This deThe WHO’s REASSURED criteria allow for the assessment of suitability of a device/method for PONT, including Real-time connectivity, Environmentally friendly, Ease of collection, Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable to end-users. In the context of sample preparation (lysis and extraction) for NA testing, the analysis focused on the criteria Environmentally friendly, Affordable, User-friendly, Rapid, Equipment-free, and Deliverable to end-users, as other criteria are strongly dependent on instrumental, amplification, and detection aspects beyond the scope of this work. Environmental sustainability of the reagents used is an emerging consideration, lowering the appeal of the traditionally popular chaotropic lysis/binding using guanidium salts owing to their acute toxicity upon direct contact and their chronic toxicity to the environment, in addition to their non-sustainable production . As mentSample preparation plays a pivotal role in the outcome of chemical/biochemical assays conducted in a laboratory or at a point-of-need setting. Sample preparation approaches developed for laboratory-based nucleic acid amplification testing (NAAT) often entail lengthy protocols and technical complexities, rendering them unsuitable for point-of-need testing (PONT). Microfluidic technologies have emerged as appealing avenues for automating workflows originally designed for benchtop assays. Concurrent with this technological change, a growing trend towards rationalising reagent usage is motivated by the desire to reduce the number of different reagents to aid process and device simplicity, the desire to minimize reagent volume to save cost, and the desire to minimize or eliminate the use of hazardous reagents to facilitate disposal.Despite encouraging results in the lysis of mammalian cells, non-chemical microfluidic lysis approaches including acoustic, piezoelectric, thermal, and electrical lysis are not as effective in the lysis of smaller and tougher microbial targets as chemical approaches. Consequently, chemical lysis has been most popular for NAAT, and a review of advances in lysis using detergents, enzymes, alkalinity, and chaotropic reagents indicates that combinations of multiple lytic agents often lead to increased lysis efficiency. The quest for reagents with decreased potential for attenuating the amplification reaction has led to viable alternatives to chaotropic agents. For example, AP lysis has enabled direct amplification from the lysate, minimising post-processing to a simple dilution to reduce the alkalinity. Solid-phase extraction, where NAs are bound to a solid support before elution into a purified aliquot for amplification, are popular, because an elution volume smaller than the sample volume can aid in concentration enhancement. Chaotropic agents can also facilitate the binding of the NA to the stationary phase during SPE. While popular in the direct translation of benchtop kits to PONT settings, concerns around the use of chaotropic agents have led to a trend away from chaotropic reagents with traditional silanol-based stationary phases towards alternate binding chemistries including the use of crowding effects and ionic binding on anionic and cationic stationary phases. The changes in binding chemistry have been accompanied with changes in the composition of binding, washing and elution buffers, for the cationic stationary phases where binding and release are facilitated by changes in pH.In recognition of a loss in NAs transferring from the solid support to the amplification environment, dual-purpose buffers have been developed that allow for elution in amplification buffer. Direct, on-bead amplification has also been presented to eliminate elution as the stationary phase remains present during amplification.The trends in NA-PONT are an increasing differentiation from standardized approaches developed for laboratory-based NAAT. In lysis, chemical approaches based on alkaline conditions or detergent/enzymatic approaches are expected to dominate because of the appeal of speed, efficiency, and the little environmental concern; new tailored mechanical lysis approaches are anticipated to be capable of quickly and effectively lysing microbial cells. In extraction, the use of chaotropic reagents is expected to further decrease because their manufacture is not sustainable; their use complicates the workflow and residues cannot be disposed with general waste. Several promising benign substitutes have been reported and are anticipated to further grow in popularity, as minimising processing steps and reagents will enhance affordability, user friendliness, and speed, while reducing equipment needs. In elution, the use of dual-purpose elution/amplification reagents as well as an increase in adoption of on-bead amplification are expected to boost sensitivity by preventing losses during elution. The workflow has been translated into a broad range of fluidic formats, with centrifugal devices dominating the field with integrated, automated processes; however, the sophisticated devices and instrumentation may cause challenges for implementation in a low-resource setting. More aligned with the WHO’s RE-EMERGED criteria are assays where immiscible barriers are used, allowing for the progression between processing steps of NAs immobilized on magnetic beads simply by dragging a magnet. Very well suited for PONT are the paper-based platforms, when the extraction and purification can be controlled using an electric field without the need for advanced fluidic control. In general, following trends in chemistry, fluidic design, and device manufacture, a growth in NA-PONT devices for disease surveillance/management is expected, meeting the growing demand for point-of-need tests (PONTs) for human and animal/plant health.


Autistic adolescents and adults commonly experience mental health concerns; however, mental health clinicians may hold implicit stigmatizing views of autism that contribute to case conceptualization and treatment goal setting that align more with caregivers’ than clients’ goals. This impingement on client autonomy is concerning, problematic, and potentially harmful for autistic clients who are of an age to set their own treatment agenda regardless of co-occurring intellectual disability and/or language delays. An application of the shared decision-making framework, an evidence-based tool for promoting client autonomy, can help to avoid these challenges in treatment. In this perspective, we use a case vignette as an anchor for discussing the imperative of honoring autistic clients’ autonomy in mental health treatment and guiding shared decision-making to reduce stigma, promote autonomy, and increase collaborative care for autistic clients in mental health treatment. Yet, In this perspective, we bring together our expertise as mental health providers and clinical researchers, as well as our lived perspectives as non-autistic neurodivergent and neurotypical allies of autistic people to discuss the imperative of honoring autistic clients’ autonomy in mental health treatment. We acknowledge that mental health clinicians may themselves be autistic or otherwise neurodivergent, and while our guidance may be helpful to all, it is most intended for neurotypical clinicians. We use a case vignette as an anchor for discussing the imperative of honoring autistic clients’ autonomy in mental health treatment and guiding decision-making. This vignette has been constructed by combining the experiences of numerous clients into one hypothetical case.Jacob, a 16 years-old Black cisgender, heterosexual young man, is autistic, has a mild intellectual disability, and is clear about what he wants from therapy. “I want to have a girlfriend,” he says. His parents tell you that that is not their goal: they want Jacob to be “less disruptive.” Jacob’s parents state that Jacob will “scream and shout” when he is upset or denied something he wants. These behaviors occur more at home than in other settings. When you try to talk to Jacob about his desire to date, his parents interject to tell you that Jacob does not need a girlfriend since, “it wouldn’t be appropriate for him to date.” In a one-on-one session, Jacob tells you that he is “really excited” to have sex, but when he got condoms from his doctor (following a conversation that Jacob initiated about safe sex), his parents took them away, saying pre-marital sex is a sin in their Christian faith.This scenario forces us to confront the complex issue of client autonomy when caregiver and client goals conflict. Jacob’s parents are focused on his “disruptive” behavior. To Jacob, having a girlfriend is a meaningful goal. Many questions come to mind when considering how to address this conflict. How do we conceptualize the presenting concerns? With whose goals do we align? Who decides what therapy goals are appropriate and legitimate? What happens when an adolescent client and their caregivers disagree on goals for therapy?We propose that supporting autonomy through the evidence-based framework of shared decision-making –8 can gu2.against promoting autonomy in therapeutic relationships ]Jacob’s clinician laid out the conflict between Jacob and his parents and the fact that treatment goals were needed; Jacob and his parents agreed this was true and that they all wanted something to change.The clinician then explored with Jacob and his parents the meaning of their goals; Jacob’s parents were able to discuss their worries for Jacob’s future and how his outbursts lead them to fear for his safety, particularly as Jacob is a Black male. Jacob needed the context of this worry explained, but when it was, he began to understand his parents’ fears.Jacob was likewise able to tell his parents he had always dreamed of being “married like you” which touched Jacob’s parents deeply. Jacob likewise explained that he knew, from school, that sex was important to do “right,” and that safe sex was “right sex.” Jacob’s parents were able to see the importance of dating to Jacob. They expressed the preference that Jacob wait to have sex until marriage, but also stated they were glad he was “thinking about safety.” Jacob, for his part, was able to agree that less conflict at home would be good.Jacob and his parents negotiated an agreement: Jacob and his parents would work with the clinician to reduce conflict at home. Jacob’s parents agreed that Jacob could date if he found a girlfriend.The end treatment goal prioritized reducing conflict. Jacob, understanding his parents’ fears for his life, was more willing to work on reducing outbursts by using his coping skills, saying “I don’t want to get shot.” The treatment goals were reviewed after six months; at that time, Jacob’s parents reported “less than one outburst a week” and Jacob was planning to ask a girl from his class to a church picnic.Furthermore, it is important to understand the role of identity in challenges to client autonomy. For Jacob, his various intersecting identities as a Black, cisgender, heterosexual autistic teen with an intellectual disability from a family with a religious background affect the way in which he is perceived and how his family or clinicians choose to interpret his behaviors. Historically, being autistic has been cited as a reason for excluding clients from shared decision-making regarding their care e.g., , 15]. In. In15)].The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.AM: Conceptualization, Writing – original draft, Writing – review & editing. KG: Conceptualization, Writing – original draft, Writing – review & editing. SS: Writing – original draft, Writing – review & editing. KC: Conceptualization, Writing – original draft, Writing – review & editing.


IntroductionDopamine is an important neuroregulatory hormone and is secreted during exercise. Its role in physiological regulation is not fully uncovered. Recent studies showed that it suppresses inflammation. Colon cancer is one of the most predominant cancers in the population and is influenced by prolonged inflammation. The anti-inflammatory effect of dopamine using the colon cancer model was analyzed in KB cells.Methods and Inhibitory Concentration- 50 (IC50) was determined by 3--2,5 diphenyl tetrazolium bromide assay. BCl-2, tumour necrosis factor-α (TNF-α), nuclear factor kappa- B (NF-kB), and interleukin (IL)-6 were assessed using reverse transcription polymerase chain reaction (RT-PCR)(at 50 and 100 µg/ ml < IC50). Schrödinger was used for docking analysis using nuclear factor Kappa B (NF-kB) (Protein Data Bank: 5T8O) and dopamine (CID 681).KB cells were cultured using Dulbecco's Modified Eagle MediumResults50- 225±3.1µg/ ml). It downregulated the expression of BCl-2, NF-k, and IL-6, but increased TNF-α expression. Dopamine bonded with NF-kB by two hydrogen bonds with aspartic acid-53and alanine-54, respectively).Results were represented as mean ± standard deviation and statistically evaluated. Dopamine showed severe growth inhibition in KB cells (ICConclusion-kB pathways in KB cells.The present study revealed that dopamine has a significant anti-cancer potential by blocking NF The messenger ribonucleic acid (mRNA) expression levels of each gene were normalized to the RNA of glyceraldehyde 3-phosphate dehydrogenase (GAPDH)using the cycle threshold (2CT) method. The primers were produced by Shanghai, China-based Invitrogen [After being exposed to dopamine for 48 hours, KB cells were treated with the TRIzol reagent to isolate the total RNA from them . Using a Prime Script RT Reagent Kit , for synthesizing, complementary deoxyribonucleic acid (cDNA) was reverse-transcribed. For the amplification, an Mx3005P Real-Time Polymerase Chain Reaction System was employed , and the kit's directions were followed for the quantitative polymerase chain reaction (Q-PCR)vitrogen .Molecular docking analysisThe structure of dopamine (PubChem database with CID 681) and NF-kB (Protein Data Base (PDB:5T8O) were retrieved. The Schrödinger software suite  was used to prepare the Epik (rapid and robust pKa predictions) states and to optimize the ligand. The docking was carried out using the extra precision method (XP) and the Glide score  was calculated and expressed as kcal/mol .Statistical analysisThe significant value was confirmed using one-way (Newman-Keuls post hoc test) and two-way ANOVA (Bonferroni post hoc test).One of the primary factors in the development of cancer is persistent inflammation. Its involvement in the development and spread of colon malignancies has been thoroughly investigated. Dopamine inhibited inflammatory effects on KB cells. On KB cells, dopamine decreased the expression of inflammatory genes such as TNF-α, NF-B, and p38 but enhanced the expression of Bax.Effect of dopamine on cell viability50 values of doxorubicin and dopamine were calculated as 125.41±1.56 and 225±3.1 µg/ml, respectively. The expression of pro and anti-inflammatory genes was analyzed in two concentrations, 50 and 100 µg/ml, respectively.Using the MTT assay, the cytotoxic effect of dopamine was investigated. For the investigation, various concentrations, such as 0, 50, 100, 250, and 500 µg/ml were employed. In comparison to controls, the findings demonstrated that dopamine exhibited dose-dependent cytotoxicity with KB cells  in the mRNA expression of TNF-α. Additionally, at a dose of 50 µg/mll Figure .Dopamine inhibited NF-kB mRNA expression on the KB cellsThe ratio of the NF-kB in KB cells treated with dopamine was analyzed using qPCR. The expression was in a dose-dependent manner. At 50 µg/ml, dopamine showed a significantly reduced NF-kB expression compared to the control Figure .Our results indicated that dopamine decreased the inflammation by attenuating the pro-inflammatory markers such as NF-kB and BCl-2. Meanwhile, it increased the expression of TGF-α (T cell growth factor - α) in a dose-dependent manner.Molecular dockingThe protein structure PDB: 5T8O), which contains the structure of murine NF-kB and kinase-bound imidazolbenzoxepin molecule, was downloaded from the PDB databank  and inhibited the protein kinase A/protein 38 (PKA/p38) signaling by activating the dopamine D2-like receptor-4 (DRD4) receptor. DRD4 is one of the dopamine family receptors (DRD1-DRD5). Thus, it inhibited the activation of tumor-associated macrophages also . DopaminOur findings further imply that dopamine might be an endogenous bioactive substance with anti-inflammatory qualities, making it a potential treatment for malignant colon cancer and other pathological disorders caused by inflammation through NF-κB signal.


Knowledge of clinical and laboratory differences between chromosomal and undefined causes aids etiological research on non-obstructive azoospermia.Compare clinical and laboratory differences between men with non-obstructive azoospermia due to chromosomal anomalies versus undefined causesA cross-sectional retrospective study conducted at a public university hospital in Campinas (Brazil)All men aged 20–40 years with non-obstructive azoospermia were included in the analysis.NR5A1 gene mutation, and 1 mild androgen insensitivity syndrome) (4%), 9 with other non-obstructive azoospermia etiologies (8%), and 79 with undefined causes. The 22 chromosomal anomaly cases  were compared with the 79 undefined cause cases. The KS group had lower average testicular volume, shorter penile length, and lower total testosterone levels but greater height, arm span, serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels, and gynecomastia frequency . Patients with testicular XX DSD had LH, FSH, and penile length data intermediate between the KS and undefined cause groups, testicular volume similar to the KS group, and other data similar to the undefined group.The 107 cases included 14 with Klinefelter syndrome (KS) (13%), 1 with mosaic KS, 4 with sex development disorders (2 testicular XX, 1 Clinical and laboratory data differentiate men with non-obstructive azoospermia and chromosomal anomalies, particularly KS and testicular XX, from those with undefined causes or other chromosomal anomalies. A study of a North American population revealed that 12% of male individuals aged 15–44 years are infertile. The main factors related to infertility include obesity, infection, neoplasms, cryptorchidism, smoking, varicocele, chromosomal anomalies, sperm duct defects, scrotal exposure to high temperatures, hormonal imbalances, celiac disease, medications, heavy metal poisoning, and exposure to ionizing radiation.Male infertility is defined as the biological inability of a man to induce pregnancy in a fertile woman after unprotected sexual intercourse for at least one year. Some cases of male infertility may also be related to disorders of sex development (DSD), such as Klinefelter syndrome (KS), testicular 46,XX DSD, and disorders related to the synthesis or action of testicular hormones.Regarding the genetic causes of male infertility, approximately 15–20% of men with severe non-obstructive azoospermia or oligospermia have microdeletions on the long arm of the Y chromosome  where the spermatogenesis genes are located. In contrast, studies of genetic causes of male infertility have mainly focused on chromosomal anomalies and Yq microdeletions. In these studies, as well as in the guidelines on male infertility, DSD are not specifically considered a cause.Currently available DSD cohorts in the literature mostly include pediatric patients, with genital ambiguity being the main reason for referral.A recent study by our group of 84 men with non-obstructive infertility (azoospermia or severe oligospermia) showed that 10 (12%) had KS, 1 had testicular DSD 46,XX, and 1 had mild androgen insensitivity syndrome (MAIS). The same study also observed 4 cases of structural anomalies of the Y chromosome, 2 Yq microdeletions, and 1 autosomal anomaly corresponding to 22% of the evaluated cases. Of total patients studied, only 14 had increased serum FSH levels, 23 had increased FSH and LH levels, and 13 had decreased testosterone levels.Considering that about one-fourth of men with severe infertility have chromosomal anomalies and that the clinical and laboratory characteristics of these men, compared to those without chromosomal anomalies, have been insufficiently studied in the literature, it is necessary to verify whether these data can be useful for distinguishing men with chromosomal anomalies from those without a clear cause of their non-obstructive azoospermia.To compare clinical and laboratory data of men with non-obstructive azoospermia due to chromosomal anomalies versus undefined causes.This observational cross-sectional retrospective study was based on a medical record analysis. All included patients received medical care from the Interdisciplinary Group for the Study of Sex Determination and Differentiation  at the Hospital das Clínicas da Universidade Estadual de Campinas (HC-UNICAMP), in Campinas, Sao Paulo, Brazil. This study included all men aged 20–40 years with non-obstructive azoospermia (absence of sperm in the ejaculate secondary to impaired spermatogenesis) who were referred to our outpatient clinic for etiological clarification between January 2010 and December 2019. The inclusion criteria were non-obstructive azoospermia (confirmed by at least two sperm counts) and no history of medication use or a disease recognized as a possible cause of male infertility. The exclusion criterion was incomplete clinical and/or laboratory data in the medical records. The project was approved by the Institution's Research Ethics Committee (CAAE: 31480020.0.0000.5404) on June 2, 2020. arm span (cm) and ratio to height, body mass index (BMI) in kg/m2, penile length (in cm), and testicular volume (in mL).The following clinical data were obtained from the patients' medical records: age (years), auto-declared race , family history , educational status , height (cm), difference from average family target height,In all cases, a conventional G-banding karyotype study was performed with 400-band resolution and a minimum count of 20 metaphases. In those with normal karyotypes, analysis of a Yq microdeletion was performed using the polymerase chain reaction multiplex technique and 28 molecular markers that mapped the three regions considered the azoospermia locus .The following laboratory data were also obtained from the medical records: LH (IU/L), FSH (IU/L), and total testosterone (ng/mL) serum concentrations, karyotype data, Yq microdeletion data, and other cytogenetic or molecular tests.The statistical analysis was performed using SPSS , version 20.0, Chicago, USA applying absolute and relative frequency data using the Mann-Whitney and Fisher tests with a level of significance of P < 0.05.FSHR gene, 1 with MAIS and confirmed mutation in the AR gene, 2 with testicular 46,XX DSD, 1 with mutation in the NR5A1 gene, and 79 with undefined cause. In the defined cause group, the cases of KS, mosaic KS, MAIS, NR5A1 mutation, and testicular XX were considered DSD , with chromosomal anomalies in KS, mosaic KS, testicular XX, and other autosomal or sex chromosome anomalies (22 of 107 [20.6%]). Of these 107 cases, 101 were included in this study, of which 79 had undefined causes and 22 had chromosomal anomalies  were excluded. Among the 101 cases, the mean age was 30.4 years ; the auto-declared race was white for 67, brown for 20, and black for 15; and the educational status was illiterate for 11, completed elementary and middle school for 53; completed high school for 27, and undergraduate school for 10. No characteristics  differed between those with KS, mosaic KS, sex chromosome and autosomal anomalies, testicular XX, and undefined etiologies. Obesity was present in 42 patients, although the data did not differ between the groups.A total of 150 cases were evaluated, but only 107 met the inclusion criteria: 14 with KS, 1 with mosaic KS , 4 with other sex chromosome anomalies , 1 with autosomal anomaly , 2 with Yq microdeletion, 2 with autosomal recessive infertility with increased FSH and no mutation in the nomalies . PatientDue to the small number of cases in some of these groups, we decided to compare only the data between the 14 patients with KS and 79 patients in the undefined cause group.There was no statistically significant difference (Mann-Whitney test) between the KS group (n = 14) and the undefined group (n = 79) in parental height (P = 0.37), arm span and height ratio (P = 0.98), or BMI (P = 0.15) . HoweverThe mosaic KS patient exhibited laboratory data  like those of the undefined group as well as average testicular volume, penile length, height, arm span, and BMI, like the KS group . PatientThis study determined that approximately 20% of non-obstructive azoospermia cases were associated with chromosomal anomalies or DSD. These results correspond with the percentage reported in the current literature (approximately 15%). (1997), approximately 3% of male infertility cases are caused by KS. In the present study, 15 of 107 cases (14%) of non-obstructive azoospermia were caused by KS. However, it is important to note that only 25% of KS carriers are diagnosed during their lifetime and that most diagnoses occur in adulthood during patient infertility investigations.KS is the most frequent genetic and chromosomal cause of non-obstructive azoospermia. According to Abramsky and ChappleSRY gene translocated on one of the X chromosomes, and their phenotype is virtually identical to that of individuals with KS. In such cases, the lack of sperm production is due to the absence of other genes on the Y chromosome.Individuals with testicular XX can also be identified among patients with non-obstructive azoospermia. For the most part, these individuals have the  Other 46,XY DSD that are not associated with gonadal differentiation disorders may also go undiagnosed during childhood and have infertility as their main complaint. This is what often happens in cases of hypogonadotropic hypogonadism, milder forms of testosterone synthesis defects, and also in androgen receptor mutations with a male phenotype (MAIS) or barely noticeable genital ambiguity, in which there is a reduction in sperm production due to defects in androgenic activity. Individuals with 5-alpha-reductase type 2 deficiency may also have more pronounced androgenization of the external genitalia which goes undiagnosed in childhood but present as adulthood infertility due to underdevelopment of the prostate and seminal vesicles. In the present study, apart from the KS and testicular XX cases, 1 case of XY partial gonadal dysgenesis due to the NR5A1 gene mutation and 1 case of MAIS with the AR gene mutation were diagnosed. Therefore, the results of the present study show that observation of clinical and laboratory data is very important for the differentiation of cases of non-obstructive azoospermia with chromosomal anomalies or DSD of other etiologies.Other autosomal or sex chromosome anomalies can also occur but at a much lower frequency, as observed in the present study. Cases of XY partial gonadal dysgenesis, combined gonadal dysgenesis, and ovotesticular DSD can also be found among individuals with typical or highly virilized male genitalia, which is only detected during adulthood infertility investigations. However, compared to undefined cases, KS patients presented with smaller testicular volume, shorter penis length, taller stature, greater arm span, higher serum LH and FSH concentrations, and lower testosterone levels. Gynecomastia was absent in the undefined cause group and was present in more than half of the patients with KS. In the literature, testicular volumes lower than 6 mL were present in more than 95% of KS cases, the same being true for increased serum gonadotropins. Decreased serum testosterone levels were present in 63–85% of KS cases and occurred more frequently in the older age group. Gynecomastia is reportedly present in 38–75% of affected adolescents and adults. Increased stature occurs in approximately 30% of children and adults with KS. Smaller penile size is present in 10–25% of children and adults with KS. Therefore, although the findings described in the present study are frequent in KS, our data confirm that they are an important tool for differentiating patients with KS from those with other non-chromosomal cases of non-obstructive azoospermia.Except for the KS and testicular XX cases, the other sex chromosomes and autosomal anomalies did not exhibit clinical or laboratory differences from cases of undefined cause. Few studies in the literature have shown these results.The patient with mosaic KS had a laboratory profile similar to that of the undefined group but had clinical features similar to KS with the exception of normal testicular volume. Previous studies demonstrated that it is difficult to pinpoint a specific clinical feature in cases of sex chromosome mosaicism without actually knowing the percentage of each lineage in various tissues, including gonads.Patients with testicular XX DSD (n = 2) exhibited clinical features similar to those of the undefined group except for decreased testicular volume and hypergonadotropic hypogonadism, but these were not as severe as the results seen in the KS group. Similar data have been described in the literature.Despite the fact that this study has all the limitations of retrospective data collection, it provides important clinical information that supports medical investigations in men with non-obstructive infertility from a significant sample evaluated homogeneously by a single clinical service over a 10-year period with karyotyping performed in all cases and Yq microdeletions investigated in all patients with a normal karyotype.Therefore, two main conclusions may be drawn from this study: first, chromosomal anomalies were the cause of approximately 20% of non-obstructive azoospermia cases. Second, clinical and laboratory differences existed among different non-obstructive azoospermia etiologies, especially undefined, KS, and testicular XX cases. For this reason, the results of this study provide an important information resource that will be very useful for physicians and other healthcare professionals during investigations and requests for complementary tests during the etiological evaluation of non-obstructive azoospermia.