A comprehensive understanding of the cerebrospinal fluid (CSF) system is essential for our understanding of health and disease within the central nervous system (CNS). The system of CSF refers to all components involved in CSF production, movement, and absorption. In recent years, extensive research has resulted in vastly improved understanding of the CSF system in health and disease. Yet, several aspects remain to be fully clarified, notably along the spinal cord as the preponderance of research has focused on the brain. This review briefly summarizes the CSF system and its implications for CNS diseases and highlights the knowledge gaps that require further research. Renewed attention has come to the cerebrospinal fluid (CSF) system due to its importance for central nervous system (CNS) homeostasis. The CSF system constitutes a crucial role in the CNS as it provides mechanical protection, ensures homeostasis, and facilitates communication between the CNS and peripheral nervous system, lymphatic system, vascular system, and immune system  water channels  and arachnoid granulations (low pressure). The movement of CSF from the spinal subarachnoid space to the perivascular spaces and the lymph system is poorly described, although similar routes have been suggested (Chen et al., via the meningeal lymphatic vessels, nerve sheaths, and nasal cribriform plate into the deep cervical lymph nodes (Iliff et al., An essential function of the CSF system is the maintenance of CNS homeostasis. As the CNS consists of highly active metabolic regions, waste products need to be cleared. The most recently proposed mechanism for waste clearance is the highly debated glymphatic system. The available literature primarily focuses on the mechanisms within the brain; thus, the mechanisms within the spinal cord remain largely elusive. The glymphatic system is a fluid conduit defined as an astrocyte-mediated fluid exchange of CSF and ISF in the brain (Iliff et al., The existence of a lymphatic network within the meninges of the brain was first described by the Italian anatomist Paolo Mascagni in 1787; a description that recently has been translated and published (Sandrone et al., Although considerable anatomical differences exist between the brain and the spinal cord, it seems reasonable to assume that the spinal cord has a waste clearance system and a lymphatic system resembling the systems proposed for the brain. In support of this assumption studies have demonstrated the existence of spinal perivascular spaces in rats (Lam et al., Knowledge of the CSF system has significant implications for understanding diseases in the brain and the spinal cord. It is, however, of great importance to acknowledge that any alteration in the CSF system may be influenced by other factors, e.g., aging, hypertension, atherosclerosis, and sleep deprivation (Benveinste et al., A physiological hyposecretion of CSF occurs with age due to increased amounts of connective tissue between the vasculature and the epithelial cells Preston, . This phvia AQP4 could facilitate protein accumulation in the brain (Rasmussen et al., The progression of neurodegenerative diseases e.g., AD has been linked to attenuation of the waste clearance system. AD is an age-dependent disease marked by the accumulation of specific proteins, neurofibrillary tangles, and amyloidß peptide, in the brain. These proteins are proposed to be cleared by the waste clearance system, thus reduced movement of CSF from the periarterial spaces to the brain parenchyma As described previously, lymphatic vessels are essential for fluid balance and immune surveillance in tissues (Oliver et al., Disturbances in the CSF movement may influence the functioning of the waste clearance system. Thus, CNS diseases causing an obstruction in the brain or along the spinal cord, and thereby CSF movement disturbances, may promote the emergence and progress of secondary CNS diseases. This is demonstrated by the association between traumatic spinal cord injury and posttraumatic syringomyelia: a disease characterized by the formation of fluid-filled cysts within the spinal cord parenchyma (Vandertop, As the preponderance of studies investigate the mechanisms of the CSF system and its implications for neurological diseases in the brain, our understanding is sparse when it comes to the mechanisms in the spinal cord. It does, however, seem reasonable to believe that the assumptions made in the brain, to some extent, are applicable to the spinal cord. Yet, the examples above highlight the complexity of the CSF system. Therefore, research is needed to evaluate the CSF system in a more integrative view to elucidate how changes in one part of the system lead to changes in other parts of the system.Despite significant advances in our understanding of the CSF system, many processes remain to be elucidated. Notably, as the majority of studies focuson the brain, there is a significant knowledge gap regarding the spinal cord. Both the anatomical and physiological differences between the brain and spinal cord hamper the translation of findings found in the brain to the spinal cord, thus more research into the mechanisms in the spinal cord is warranted.All authors contributed to conception and design of the work. TW and HD wrote sections of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In conclusion, CSF p‐tau235 appears to be not only a highly specific biomarker of AD but also a promising staging biomarker for the preclinical phase. Thus, it could prove useful tracking disease progression and help enriching clinical trial recruitment.Alzheimer’s disease (AD) is characterised by a long preclinical phase. Although phosphorylated tau (p‐tau) species such as p‐tau217 and p‐tau231 provide accurate detection of early pathological changes, other biomarkers capable of staging disease progression during preclinical AD are still needed. Combining exploratory and targeted mass spectrometry methods in neuropathologically confirmed brain tissue, we observed that p‐tau235 is a prominent feature of AD pathology. In addition, p‐tau235 seemed to be preceded by p‐tau231, in what appeared to be a sequential phosphorylation event. To exploit its biomarker potential in cerebrospinal fluid (CSF), we developed and validated a new p‐tau235 Simoa assay. Using three clinical cohorts, we demonstrated that (i) CSF p‐235 increases early in AD  A combination of mass spectrometry analysis of brain tissue and ultrasensitive immunoassays in three independent CSF cohorts identifies p‐tau235 as a novel biomarker for Alzheimer's disease (AD) – from preclinical stages to dementia. Despite the success in the development of fluid biomarkers to detect Alzheimer’s disease (AD) pathology, there remains a need for biomarkers to characterise the long preclinical stage of the disease. It is becoming clear in the AD field that a detailed understanding of the preclinical stage is essential to develop primary and secondary strategies to prevent this fatal disease. Currently, highly accurate biomarkers are available to detect preclinical AD . However, these biomarkers are not optimal in determining if an individual is at the beginning of this 15–20 year preclinical period or imminently progressing to cognitive impairment.We first examined neuropathologically confirmed AD and control brain tissue and employed a phosphoproteomic mass spectrometry method which highlighted the prominence of p‐tau235 in AD pathology. Our findings strongly suggest that threonine 231 and serine 235 undergo sequential phosphorylation as a result of AD pathology, and would pose a great potential to track disease progression. Using an in‐house developed Simoa assay specifically targeting CSF p‐tau235, we measured this p‐tau species in three independent CSF cohorts, demonstrating that CSF p‐tau235 is a highly specific novel biomarker to detect AD. Moreover, we show the predominance of the double‐positive p‐tau231/235 in individuals classified as late‐stage preclinical AD, while single positivity for CSF p‐tau231 was shown to be a more defining feature of early preclinical disease.These results have great potential in the design of observational or interventional studies at the preclinical stage of the disease: a study aiming to target the earliest stage of preclinical AD would include single positive p‐tau231 participants, whereas a study in asymptomatic individuals but closer to symptom onset would include double‐positive p‐tau231/235 participants. In trials evaluating disease‐modifying drugs, which are increasingly focusing on the preclinical stage of AD, p‐tau235 could be used as an inclusion or exclusion biomarker depending on the preclinical stage being targeted.et al, et al, et al, et al, et al, 1–42 (or Aβ1–42/40 ratio), phosphorylated tau at threonine 181 (p‐tau181) and total tau (t‐tau)   report estimated that 50 million people worldwide suffered from dementia and that the number of cases would triple by 2050, which is paralleled by an increase in the medical costs estimated as 2 trillion US$ by 2030. AD emerges as the most prevalent cause of dementia, accounting for 50–60% of all cases  and antibody‐based methods have greatly contributed to our knowledge regarding the time course of different p‐tau species in fluids, helping to further understand their potential as biomarkers of AD pathology during disease progression , although it displayed a moderate increasing trend   soluble fraction of human cortical brain homogenates. We used the TBS‐soluble material because the CSF composition partially reflects this fraction  Fig . Additio001) Fig . Thus, oP < 0.0001)   and positive correlations with t‐tau  and p‐tau181   Fig . CSF p‐t.00) Fig . In addiP < 0.0001), cognitively unimpaired Aβ pathology negative  and cognitively unimpaired Aβ pathology positive . Similarly, CSF p‐tau235 was significantly increased in mild cognitive impairment due to AD (Aβ pathology positive) (MCI+) when compared with non‐AD (P < 0.0001), CU− (P < 0.0001) and CU+ (P < 0.0001) , which indicates that CSF p‐tau235 emerges very early in the AD continuum, specifically during preclinical stages , CU−  and CU+  . In the TRIAD cohort, CSF p‐tau235 was significantly increased in AD when compared with non‐AD (001) Fig . We alsoages Fig . CSF p‐t.96) Fig .235 = 0.73, CI95% = 0.61–0.85) was significantly lower than that of CSF p‐tau231 , but not significantly different from that of CSF p‐tau217    and p‐tau231  (Appendix Table rS = 0.66 P < 0.0001) and tau PET  in the whole cohort. Within the respective groups of PET‐positive cases, CSF p‐tau235 correlated slightly better with the tau PET  than with the Aβ PET  , followed by a very prominent increase between Braak I/II and III/IV (P < 0.0026). No significant differences could be found between Braak III/IV and V/VI (P = 1.0), although CSF p‐tau235 appeared to display an increasing trend  Fig . In cont.66) Fig . In addi235 = 0.49, P < 0.0001) . In contrast, CSF p‐tau235 displayed a prominent increase between the A+T− and A+T+ groups  . We performed ROC analyses to determine the accuracy of CSF p‐tau235 to discriminate between the different AT groups and compared it with that of CSF p‐tau217 and p‐tau231    but was not better than CSF p‐tau231   of Aβ (A) and tau pathology (T) in CSF into three groups: A−T−, A+T− and A+T+. Thus, we leverage the unique characteristics of the ALFA+ cohort to study CSF p‐tau235 in preclinical AD. We found that CSF p‐tau235 was mildly, but significantly, increased in the A + T− when compared with the A−T− group (Cohen’s d (d)001) Fig , further001) Fig , whereasu231 Fig . Both CS001) Fig . Yet, CS073) Fig . We also.18) Fig .rS = 0.32, P < 0.0001)  and correlated strongly with p‐tau231 , highlighting the close relationship between p‐tau235 and p‐tau231 during early AD stages , followed by CSF p‐tau217 and finally p‐tau235  of the A−T− group as a cut‐off, all individuals were classified as [231−/235−], [231+/235−], [231−/235+] and [231+/235+] and plotted in a stacked bar chart in order to illustrate the sequence of phosphorylation in CSF Fig . In the et al, In this study, we report a novel biomarker discovery, p‐tau235, starting from its quantitative assessment in AD brain tissue, followed by immunoassay development and quantification in CSF. By the combined use of exploratory and targeted IP‐MS approaches, we were able to identify and quantify a double‐phosphorylated tau species containing p‐tau235 in human brain tissue, which we demonstrated to be highly increased in AD. In order to translate these findings from the brain into CSF, we developed a Simoa assay specifically targeting p‐tau235. Subsequently, we measured CSF p‐tau235 in three independent cohorts and demonstrated that (i) CSF p‐tau235 has a high diagnostic accuracy to identify symptomatic AD, similar to CSF p‐tau217 and p‐tau231; (ii) CSF p‐tau235 is significantly increased early in preclinical AD, when only subtle changes in Aβ pathology are detectable in CSF; (iii) CSF p‐tau235 matched the performance of CSF p‐tau231 and outperformed CSF p‐tau217 when discriminating preclinical groups (A+T− vs A+T+); (iv) during the preclinical stage of AD, there is a sequence of changes in p‐tau biomarkers consisting on an initial increase in CSF p‐tau231, followed by an increase in CSF p‐tau235. These changes in CSF are consistent with the sequential phosphorylation described by us in TBS‐soluble AD brain fractions and by others in insoluble PHF material  and then plateau at later stages (only a subtle and not significant increase occurred between MCI+ and AD dementia). This plateau is similar to what has been found for CSF p‐tau217 and p‐tau231 . CSF p‐tau217, on the other hand, was not significantly different from CSF p‐tau235 in this scenario, but its AUC value was higher. This suggests that CSF p‐tau217, similarly to CSF p‐tau231, is superior to CSF p‐tau235 in capturing the earliest AD changes. In contrast, CSF p‐tau235 had a similar accuracy to CSF p‐tau217 and p‐tau231 distinguishing CI− and CI+, which indicates that once cognitive impairment is clinically established, all p‐tau assays perform similarly identifying AD pathology. Secondly, when examining preclinical AD stages in more depth in the ALFA+ cohort, we demonstrated that CSF p‐tau217 and p‐tau231 outperformed CSF p‐tau235 to discriminate A+T− from A−T− presumably because both p‐tau217 and p‐tau231 increase slightly earlier in preclinical AD. Conversely, CSF p‐tau235 had similar accuracies distinguishing A+T+ from A−T− when compared with CSF p‐tau217 and p‐tau231. This again demonstrates that all p‐tau assays perform similarly in identifying AD pathology, if both amyloid and tau pathologies are present. Remarkably, when discriminating between preclinical groups (A+T− vs A+T+), CSF p‐tau235 matched the performance of CSF p‐tau231 and outperformed CSF p‐tau217. Further examination of these data revealed the effect size (as determined by Cohen’s d) of the increase in CSF p‐tau235 between A+T− and A+T+ was indeed larger than that of CSF p‐tau231 and p‐tau217. Finally, using Aβ PET as a proxy of disease progression, we modelled the trajectory of CSF p‐tau235 in preclinical AD, situating the emergence of abnormal CSF p‐tau235 values after CSF p‐tau231 and p‐tau217 and before CSF p‐tau181 and total tau. Thirdly, we demonstrated that CSF p‐tau231 and p‐tau235 positivity occurs in a sequential manner during preclinical AD, that is, there is a gradual increase in these phosphorylations across the A−T−, A+T− and A+T+ groups. Importantly, most of the CSF p‐tau235‐positive cases were already CSF p‐tau231 positive, but the opposite was not observed. While 374 out of 383 (97.65%) of the cases concord with the sequential phosphorylation hypothesis in CSF, there were only 9 out of 383  discordant cases. These results suggest that the neurpathological findings of a sequential phosphorylation affecting threonine 231 and serine 235 residues in insoluble PHFs . We show here that p‐tau235 is a novel and highly specific CSF biomarker for different stages of AD, including preclinical AD. Moreover, combined pathological and CSF data, independently assessed by MS and immunoassays, suggest that the combination of CSF p‐tau235 and p‐tau231 biomarkers may be useful not only to detect preclinical AD but also to stage this long asymptomatic period. Such characterisation could greatly benefit future drug development in clinical trial recruitment, finding the best window to assess the efficacy of candidate compounds or simply evaluating if disease progression has been effectively tackled.In conclusion, this is the first study to demonstrate the role of p‐tau235 with AD in brain tissue and translate this finding into clinical utility, by the detection of p‐tau235 in CSF from individuals at different stages of the AD et al, et al, et al, et al, et al, et al, et al, Human post‐mortem brain tissue from frontal grey matter of neuropathology confirmed sporadic AD and control cases was obtained through the brain donation programme at Queen Square Brain Bank for Neurological Disorders (QSBB), Department of Clinical and Movement Neurosciences, Institute of Neurology, University College London (UCL). Sample description and case demographics are presented in Appendix Table Diagnostic performance of the developed Simoa p‐tau235 was evaluated in three different research cohorts:n = 19) and neurological controls  . Individuals with other neurological conditions such as cerebrovascular or co‐existing inflammatory disease were excluded. Control individuals consisted of patients with normal core AD CSF biomarkers levels and minor psychiatric and neurological symptoms. Ethical approval for research use of these samples has been granted by the Ethics Committee at the University of Gothenburg (EPN 140811).The discovery cohort consisted of CSF samples from AD patients  (Table n = 50), cognitively unimpaired Aβ pathology positive , mild cognitive impairment due to AD (Aβ pathology positive) , AD (n = 20) and non‐AD (n = 19). This last group included MCI patients negative for Aβ pathology and those clinically diagnosed with frontotemporal dementia. CU participants scored 0 on the Clinical Dementia Rating (CDR) scale, whereas MCI had a CDR score 0.5 and AD had a CDR score greater than 1. MCI patients also presented objective and subjective memory impairments but essentially normal daily living activities. The diagnosis of AD was given according to the National Institute on Aging and the Alzheimer’s Association criteria for probable AD  and cognitively impaired Aβ pathology negative  in order to analyse the performance of CSF p‐tau235 once cognitive impairment is established. The TRIAD study was approved by the Research Ethics Board of the Montreal Neurological Institute and the Faculty of Medicine Research Ethics Office . All study participants provided written informed consent.The cross‐sectional samples reported here belong to the Translational Biomarkers of Aging and Dementia (TRIAD) cohort  and were stratified based on amyloid pathology using CSF AβAPOE status, verbal episodic memory score and CAIDE score were taken into consideration. A detailed phenotyping was performed in ALFA+ participants, including a lumbar puncture for the measurement of CSF biomarkers and imaging (MRI and PET) biomarker acquisition. ALFA+ inclusion criteria were (i) individuals who had previously participated in the 45–65/FPM2012 study (ALFA parent cohort); (ii) age between 45 and 65 years at the time of inclusion in the cohort (45–65/FPM2012 study); and (iii) long‐term commitment to the study: inclusion and follow‐up visits and agreement to undergo all tests and study procedures . The ALFA+ exclusion criteria were (i) cognitive impairment ; (ii) any significant systemic illness or unstable medical condition which could lead to difficulty complying with the protocol; (iii) any contraindication to any test or procedure; and (iv) a family history of monogenic AD. Participants in ALFA+ were stratified based on the presence of Aβ pathology (A+) CSF Aβ1–42/40 < 0.071) and tau pathology  on an automated e 601 Cobas instrument   study includes 2.743 middle‐aged (45–74 years old) cognitively unimpaired individuals enriched for a family history of AD. The nested longitudinal ALFA+ study includes 450 participants selected based on their specific AD risk profile, determined by an algorithm in which participants with an AD parental history and et al, Protein extraction from human brain tissue has been previously described in detail , 4 μg of the monoclonal antibody anti‐tau HT7  was conjugated with 50 μl M‐280 Dynabeads  according to the manufacturer’s protocol. HT7‐coated beads were used to immunoprecipitate brain extracts containing 10 μg of total protein. Every sample was brought to a final volume of 1 ml with phosphate‐buffered saline  buffer, adding Triton X‐100 to a final concentration of 0.05%, and subsequently incubated overnight at +4°C. Immunoprecipitation from pooled CSF  was performed using the same aforementioned parameters but with minor protocol modifications. Monoclonal Tau12  or polyclonal anti p‐tau235 antibodies were conjugated with M‐280 Dynabeads . The conjugated beads were added directly to 10 ml of pooled CSF, and every sample was brought to a final concentration of 0.05% Triton X‐100. The samples were incubated overnight at +4°C. In both cases (brain and CSF samples) after incubation, the beads and samples were transferred to a KingFisher magnetic particle processor (ThermoFisher Scientific) for automatic washing and subsequent elution of the bound protein. Elution was achieved with 100 μl of 0.5% formic acid. Eluates were collected, dried in a vacuum centrifuge and stored at −80°C until further use.220TREPKKVAVVR[T]PPKSPSSA(K)SRLQT245 and 220TREPKKVAVVR[T]PPK[S]PSSA(K)SRLQT245, containing one or two phosphorylations . The K240 amino acid was labelled with 13C and 15N for both peptides as indicated (K). To take into account the efficiency of trypsin digestion, the standards extend five amino acids both N and C terminally of the monitored tryptic peptide, 225KVAVVRTPPKSPSSAK240.For the quantitative analysis of targeted IP‐MS in individual TBS‐soluble fractions, 100 fmol of stable isotope‐labelled phosphorylated peptide standards (ThermoFisher Scientific) were added directly to each individual sample. The peptide standards consisted of Both individual and pooled TBS‐soluble brain fractions were trypsin‐digested prior to MS analysis, using 100 ng of trypsin (Promega) in 50 mM ammonium bicarbonate. The samples were brought to a final volume of 50 μl with ammonium bicarbonate and shaken overnight at 100 RPM at +37°C. Trypsination was stopped by adding formic acid to a final concentration of 1%. The samples were dried in a vacuum centrifuge and stored at −80°C pending analysis. No standards were added to immunoprecipitated pooled CSF, which was analysed either trypsin‐digested as previously mentioned or without trypsin (in order to identify endogenous peptides).et al, 6, injection time 250 ms; for MS/MS the normalised collision energy was set at 25. For the quantitative analysis, an inclusion list was used to ensure identification of the ions of interest. LC‐MS data were searched against a custom‐made tau database using Mascot Deamon v2.6/Mascot Distiller v2.6.3 (Matrix Science) for charge and isotope deconvolution prior to the search using the Mascot search engine; see for  with an in‐house developed immunoassay using a Simoa platform on an HD‐X instrument (Quanterix) at the Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden. CSF p‐tau235 assay validation is described in detailed in 18F]AZD4694) and a tau ([18F]MK‐6240) PET scan on a Siemens High Resolution Research Tomograph , followed by a T1‐weighted imaging acquisition on a 3T MRI scanner for co‐registration purposes, as previously described . Individual Braak stage classification was done, as described elsewhere  PET scan and a T1‐weighted MRI scan acquired within one year of the CSF determinations (82 ± 181 days). A high‐resolution 3D T1‐weighted MRI sequence was acquired in a 3T Philips Ingenia CX scanner . PET scans were acquired in a Siemens Biograph mCT scanner  for 90 min after the administration of a bolus of 185 MBq of [18F]flutemetamol, following a cranial computed tomography scan for attenuation correction. PET data were acquired for 20 min, using 4 frames of 5 min, and images were reconstructed in 4 frames × 5 min using 3D ordered subset expectation maximisation algorithm  by incorporating time of flight and point spread function modelling. PET images were processed following a validated Centiloid pipeline using SPM12  and one‐way ANOVA adjusted by age and sex, followed by Bonferroni‐corrected post‐hoc pairwise comparisons (multiple categories). The size effect of group differences was determined using Cohen d (d) adjusted by age and sex. Associations between biomarkers were assessed using Spearman’s rank correlation (rS). The specificity and sensitivity of p‐tau235 assay were determined based on the area under the curve (AUC) of receiver operating characteristics (ROC) analysis. The 95% confidence interval of AUC is abbreviated as CI95% . Comparisons between AUC values of different biomarkers were performed using DeLong test package . Sequential phosphorylation hypothesis involving p‐tau231 and p‐tau235 in CSF was evaluated using two different cut‐off methods to determine biomarker positivity/negativity: (i) mean ± 2 SD and (ii) 95th percentile, both calculated in A‐T‐ group (ALFA+). The cut‐offs were subsequently used for defining the samples into four categories: [231−/235−], [231+/235−], [231−/235+] and [231+/235+]. [231−/235+] cases were considered discordant with the sequential phosphorylation hypothesis , MedCalc  and R programming language were used for statistical analysis. Visualisations were generated with GraphPad Prism . Box plots display the median and the interquartile range; whiskers show the 25JL‐R, AS, GB, MS‐C, PRN, HZ and KB created the concept and design. Data acquisition was performed by JL‐R, AS, EC, LG‐M and GB. JL‐R, AS, AB, MM‐A, GB and MS‐C performed data analysis. JL‐R, AS, ALB, MM‐A, EC, LM‐G, NJA, AV, TKK, JDG, GS, MS, CET, TLL, HZ, MS‐C, GB, PRN and KB contributed to sample selection and/or interpretation of data. JL‐R and AS drafted the manuscript, and all authors revised it. All authors read and approved the final manuscript.HZ has served at scientific advisory boards and/or as a consultant for Alector, Eisai, Denali, Roche Diagnostics, Wave, Samumed, Siemens Healthineers, Pinteon Therapeutics, Nervgen, AZTherapies, CogRx and Red Abbey Labs; has given lectures in symposia sponsored by Cellectricon, Fujirebio, Alzecure and Biogen; and is a co‐founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program. KB has served as a consultant, at advisory boards, or at data monitoring committees for Abcam, Axon, Biogen, JOMDD/Shimadzu. Julius Clinical, Lilly, MagQu, Novartis, Prothena, Roche Diagnostics, and Siemens Healthineers, and is a co‐founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program. MSC has served as a consultant and at advisory boards for Roche Diagnostics International Ltd and has given lectures in symposia sponsored by Roche Diagnostics, S.L.U and Roche Farma, S.A.AppendixClick here for additional data file.


P = 0.000015). Our findings suggested that patients in the dorsal striatum group were more likely to discontinue smoking than those in the non-dorsal striatum group. The characteristics of this interruption is that smoking can be quit more easily and quickly without recurrence and that the impulse to smoke is reduced. These results suggest that the dorsal striatum is a key area for addiction to smoking.Studies have shown that addictive behavior is associated with many brain regions, such as the insula, globus pallidus, amygdala, nucleus accumbens, and midbrain dopamine system, but only a few studies have explored the role of the dorsal striatum in addictive behavior. In June 2020, we started contacting 608 patients who were hospitalized between January 2017 and December 2019, and we recruited 11 smoking addicts with dorsal striatum damage and 20 controls with brain damage that did not involve the dorsal striatum . All study participants had brain damage due to acute cerebral infarction. Disruption of smoking addiction was found to be significantly associated with the dorsal striatum (Phi = 0.794770,  Addiction is a pathological process that involves learning and memory. Potentially addictive drugs activate the brain's reward system, adaptively changing the structure and function of the nerves in this part of the brain. Research on addiction, which has increased in the past 10 years, indicates that many areas of the brain are involved in the addiction pathway, including the insula, globus pallidus, amygdala, nucleus accumbens, ventral striatum, frontostriatal, and the midbrain dopamine system  they suffered an acute cerebral infarction; (2) their lesions could be visualized using MRI; (3) they had a smoking history; (4) they did not suffer from amnesia or severe aphasia; and (5) they were not addicted to drugs other than nicotine at the time of lesion onset, per their medical records.Starting in June 2020, we attempted to contact 608 patients who met these inclusion criteria to determine their smoking history: 78 patients could not be contacted , and 157 patients reported they smoked at some time, but quit a number of years before lesion onset. Of the 373 remaining patients, 198 reported that they smoked more than 10 cigarettes per day for more than 10 years at the time of lesion onset. Among these 198 patients, 11 had only dorsal striatum damage and 20 had non-dorsal striatum damage . Each of these 31 patients was asked to complete the Fagerström Test for Nicotine Dependence (FTND), which is the most valid and commonly used scale for measuring nicotine addiction .Group differences in demographic characteristics and detailed smoking data were analyzed by SPSS Version 20.0 . Specifically, the means of the dorsal striatum group and non-dorsal striatum group were compared at baseline on the following variables: sex composition  current age, age at lesion onset, years of smoking at lesion onset, and number of cigarettes smoked per day at lesion onset. An independent two-sample The 31 patients were interviewed in order to determine how their smoking behavior changed after lesion onset (Information was obtained from relatives when necessary). All the patients were asked whether or not they had smoked in the past 6 months. Patients who reported they smoked during the past 6 months were classified as “non-quitters.” Those who reported they did not smoke during the past 6 months were classified as “quitters.” According to the classification method of Naqvi et al. , all of 2 .A 3.0 T MRI system  was used for all data collection. The head coil had a 16-channel phased-array. Other imaging parameters were as follows: T1-weighted images ; T2-weighted images ; Fluid attenuated inversion recovery (FLAIR) sequence ; Diffusion weighted imaging sequence with b-values = 1000 s/mmp > 0.05)  , 2.P = 0.000015). When we examined the right and left dorsal striatum separately, we found that the likelihood of having a disruption of smoking addiction was significantly higher after a right dorsal striatum lesion than it was after a non-dorsal striatum lesion , and it was also significantly higher after a left dorsal striatum lesion than it was after a non-dorsal striatum lesion . There were two patients who had a disruption of smoking addiction after suffering brain damage that did not involve the dorsal striatum. When examined their lesions, each of them had damage in a unique set of regions. This raises the possibility that certain patients may have a disruption of smoking addiction as a general effect of suffering brain injury.Based on the criteria described in section Behavioral Classification, 14 of the 31 patients were “non-quitters,” and 12 of the patients were “quitters” who quit smoking after lesion onset and met all four criteria for “having a disruption of smoking addiction.” The five remaining patients were “quitters” who failed to meet all four of these criteria, so they were classified as having “no disruption of smoking addiction” . More deA review of the literature shows that few studies have examined the disruption of addiction after brain injury and most of those that have are case reports . A studyThe ventral striatum has classically been considered to play an important role in addiction. Many studies have implicated the ventral striatum in the anticipation and immediate response to rewards  for the publication of any potentially identifiable images or data included in this article.ChuJ performed the statistical analysis. ChangJ drafted the manuscript. LZ, GH, JZ, LY, NS, and TZ carried out the acquisition of data. QM helped to draft the manuscript. JF conceived the study, and participated in its design and coordination. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


ASPM leading to a truncation of the ASPM protein after amino acid position 1830. The phenotype of the patients was associated with microcephaly, epilepsy, and behavioral and cognitive deficits. Despite the obvious genetic similarity, the affected patients show a considerable phenotypic heterogeneity regarding the degree of mental retardation, presence of epilepsy and MRI findings. Interestingly, the degree of mental retardation and the presence of epilepsy correlates well with the severity of abnormalities detected in brain MRI. On the other hand, we detected no evidence for substantial nonsense-mediated ASPM transcript decay in blood samples. This indicates that other factors than ASPM expression levels are relevant for the variability of structural changes in brain morphology seen in patients with primary hereditary microcephaly caused by ASPM mutations.Here, we report a consanguineous family harboring a novel homozygous frame-shift mutation in  Mentally disabled patients suffering from epilepsy, and especially from drug-resistant epilepsy, need an exact and widespread work up, including genetic testing, as clear diagnosis helps the patients and their caregivers to understand and accept the diagnosis. Furthermore, it helps the treating clinician to find appropriate treatment regimens as well as prevents initiation of useless treatments ,2.ASPM  gene mutations  are the most frequent underlying genetic cause for autosomal recessive MCPH  and and20] aASPM expression, in particular the number of IQ domains, were suggested to be correlated with cortical development and it is discussed as one of the essential genes important for the increase of the cortical size during primate evolution [ASPM, the most common recessive microcephaly gene, reduce the cortical volume by at least 50% in humans, but have little effect on the brains of mice, which probably reflects evolutionarily divergent functions of this protein. The major role of ASPM in the early cortical development and gyration has been later confirmed in Aspm knockout ferrets [ASPM transcripts leading to different cortical surface areas have been attributed to the large phenotypic variability of primary hereditary microcephaly (MCPH) patients due to ASPM mutations. This transcript variability could be caused by a different degree of nonsense-mediated decay of ASPM transcripts harboring nonsense mutations. This would explain why so far, only nonsense, splice-site or frame-shift mutations in this particular gene have been associated with autosomal recessive MCPH caused by ASPM mutations [ASPM transcript decay. Therefore, the missing non-functional protein with the appropriate amount of IQ domains  may alleviate or exacerbate the severity of the disease, resulting in the variability of phenotypic outcomes [These large phenotypic differences are unlikely to be related to differences in outcomes . EspeciaASPM mutations correlates well with the degree of structural brain abnormalities. The expression levels of the transcript for the truncated ASPM protein suggest that differences in the degree of structural cerebral changes cannot be explained by nonsense-mediated decay causing differences in ASPM expression.The large phenotypic variability seen in patients with primary hereditary microcephaly due to


Despite widespread use, the optimal implementation and clinical impact of FilmArray Meningitis Encephalitis Panel  multiplex PCR testing of cerebrospinal fluid (CSF) in children with suspected (CNS) infections is unknown.Table 1: FilmArray Meningitis Encephalitis Panel Test Characteristicsst dose of antimicrobials targeted to identified pathogen, or cessation when no treatable pathogen identified).A pre-post quasi-experimental cohort study to investigate the impact of implementing MEP using a rapid CSF diagnostic stewardship program was conducted at Children’s Hospital Colorado (CHCO). MEP was implemented with EMR indication selection to guide testing to children meeting approved use criteria: i. infants < 2mo, ii. immunocompromised, iii. encephalitis, iv. > 5 WBCs in CSF. Positive results were communicated with antimicrobial stewardship real-time decision support . All cases with CSF obtained by lumbar puncture (LP) sent to the CHCO microbiology laboratory meeting any of the 4 criteria above were included with pre-implementation controls (2015-2016) compared to post-implementation cases (2017-2018). Primary outcome was time-to-optimal antimicrobials  and pre-implementation (n=1124) group characteristics are in Table 2. Following implementation, MEP was sent in 72% of cases, largely replacing pathogen-specific singleplex CSF testing (Table 3). Time-to-optimal antimicrobials decreased by 10 hours . There were no differences in time-to-effective antimicrobials, hospital admissions, antimicrobial starts or length of stay. Time-to-positive CSF results was faster , IV antimicrobial duration was shorter  with infectious neurologic diagnoses more frequently identified . Overall, 3% had bacterial and 9% viral CNS infection identified. Enterovirus (n=128) was most common, then HSV (n=28) and parechovirus (n=17) with similar detection rates between groupsImplementation of MEP with a rapid CNS diagnostic stewardship program improved antimicrobial use with faster results shortening empiric therapy. Routine MEP testing in high-yield cases rapidly detects common viral causes and rules out bacterial targets to enable antimicrobial optimizationSamuel R. Dominguez, MD, PhD, BioFire Diagnostics DiaSorin Molecular (Consultant)Pfizer (Grant/Research Support) Samuel R. Dominguez, MD, PhD, BioFire  Involved: Self): Consultant, Research Grant or Support; DiaSorin Molecular  Involved: Self): Consultant; Pfizer  Involved: Self): Grant/Research Support


Comparison with the mono‐triarylboranes reveals the large influence of the bridging unit on the properties of the bis‐triarylboranes, especially those of the cationic compounds. Based on these preliminary investigations, the interactions of 1+Cat, 2+Cat, 2+Cat(i), and 3+Cat with DNA, RNA, and DNApore were investigated in buffered solutions. The same compounds were investigated for their ability to enter and localize within organelles of human lung carcinoma (A549) and normal lung (WI38) cells showing that not only the number of charges but also their distribution over the chromophore influences interactions and staining properties.The synthesis, photophysical, and electrochemical properties of selectively  Four selectivelymono‐,bis‐ andtris‐trimethylammonium‐substituted, bithiophene‐bridged bis‐triarylboranes were synthesized and their potential biological applications were investigated including live cell imaging and photo‐induced cell death. It is demonstrated that their photophysical and electrochemical properties and their interactions with DNAs, RNA and cells, are strongly influenced by the number and distribution of charges within the compounds. Additionally, we demonstrate that biomolecule binding and staining selectivity for these compounds depends on the number and distribution of the cationic charges. Thus, the neutral compounds Neut1‐Neut3 and the resulting selectively charged compounds 1+Cat–3+Cat 2, and Neut3 was made possible via a synthetic route to unsymmetrically‐substituted triarylboranes which was previously not well developed,The synthesis of Neut4 and 4+Cat.mono‐ and di‐halogenated bithiophenes  as well as three different, borylated triarylboranes 4b, 5b, and 6b were required , BF3 ⋅ OEt2, and 2,6‐dimethylphenyl lithium using the general approach developed by Grisdale and co‐workers.2BFMes and subsequent reaction with 2,6‐dimethylphenyl lithium gave the triarylborane 4a in 76 % yield over two steps. C−H borylation with [Ir(COD)(μ‐OMe)]2 as the precatalyst, 4,4′‐di‐tert‐butyl‐2,2′‐dipyridyl (dtbpy) as the ligand, and bis‐pinacolato diboron (B2pin2) gave 4b in 90 % yield. The 1H NMR spectrum of 4b matches that reported in the literature.3 using Pd2(dba)3 ⋅ CHCl3 as the precatalyst, 2‐dicyclohexylphosphino‐2′,6′‐dimethoxybiphenyl (SPhos) as the ligand, and potassium hydroxide as the base gave Neut0 in 31 % yield (Scheme Neut(i)2 was synthesized from 5b and 5,5’‐dibromo‐2,2’‐bithiophene 3 in 53 % yield.For the synthesis of Neut1, Neut2, and Neut3, 5‐bromo‐2,2’‐bithiophene 2 was cross‐coupled with 4b, 5b, and 6b, respectively, in Suzuki‐Miyaura reactions using the conditions described above to give 7a, 8a, and 9a in 71 %, 93 %, and 80 % yields, respectively . However, the first reaction sequence gave higher yields in all steps, especially the last one (60 % vs. 6 % yield).To obtain Neut1–3 in hand, their cationic counterparts were synthesized using methyl triflate in CH2Cl2 similar to previous reports[PF6] as the electrolyte and a scan rate of 250 mV s‐1. Each measurement is referenced to the ferrocene/ferrocenium (Fc/Fc+) couple. The redox potentials of uncharged and cationic triarylboranes 4a–6a, 5c, and 6c were examined as well as those of the neutral and cationic bithiophene bis‐triarylboranes Neut0–4 and 1+Cat–2+Cat. Potentials of the neutral compounds were measured in THF, and those of the cationic compounds in MeCN. All cyclic voltammograms are shown in the Supporting Information  together with an extended discussion thereof. The electrochemical data are summarized in Table 4a, 5c and 6c, within the electrochemical window of the solvent used. In the case of 5c and 6c, the presence of ca. 0.5 % starting material was confirmed by 1H NMR spectra. Thus, the minor oxidation waves observed for both compounds can be attributed to the presence of non‐ or partially methylated starting materials.Electrochemical measurements were performed in the respective solvent (THF or acetonitrile) with [4a, 5a, 6a, 5c, and 6c one partially reversible 1e− reduction potential for the boron center and one 1e− oxidation potential per amino group was found. The reduction potentials decrease with increasing electron density at the boron center which increases in the order 6c<5c<4a<5a<6a. The electrochemical data are in good agreement with values reported by Gabbaï and coworkers for very similar trimethylammonium‐substituted triarylboranes. However, the trend that each trimethylammonium group leads to a decrease of 0.26 V in MeCN5c vs. 6c) but not for the addition of the first one (4a vs. 5c).For the triarylboranes bis‐triarylboranes in THF  suggesting negligible delocalization between the two boron atoms. Furthermore, a second (irreversible) 1e− reduction at ca. −3.05 V was observed. For all dimethylamino‐substituted bis‐triarylboranes, an irreversible oxidation occurred at ca. 0.35 V; only Neut3 has an additional irreversible oxidation at 0.49 V.For all neutral F Figure , one parbis‐triarylboranes  and 4+Cat display only one reduction potential. These result from two simultaneous 1e− reductions (Table S20), as the two boron centers in the latter are the same on both sides of the molecule. It was not possible to improve the resolution of the quasi‐reversible reduction waves of 1+Cat by pulse voltammetry experiments .For all cationic s Figure , two fulcalc, ΔEopt, ΔECV) were obtained from DFT calculations and experimental values. The absorption spectra were used to determine the optical bandgap ΔEopt and ΔECV was calculated from the HOMO and LUMO energies according to a literature procedure (see Supporting Information).[opt does not change significantly within a series of neutral or cationic bis‐triarylboranes but decreases by ca. 0.5 eV upon introduction of the strong dimethylamino electron donor in the mono‐triarylboranes, due to destabilization of the HOMO more than the LUMO, as supported by DFT calculations. The values of ΔEopt and ΔECV are of the same magnitude and display the same trends. For the cationic compounds, only the cationic moiety was used in the calculations, so ion‐ion‐interactions were not considered.HOMO‐LUMO gaps (ΔErmation).48, 492‐saturated acetonitrile solutions was investigated for the triarylboranes 4a, 6c, and 5c as well as for the charged bis‐triarylboranes 1+Cat–4+Cat by monitoring the weak 1O2 phosphorescence emission at 1275 nm vs. that obtained from a solution of the known sensitizer perinaphthenoneThe ability to sensitize singlet oxygen in O4a, 6c, and 5c sensitize 1O2 with efficiencies of 0.3, 0.5, and 0.6, respectively,  which increases with increasing number of trimethylammonium substituents (Table S17). The corresponding fluorescence quantum yields in acetonitrile decrease in the same order from 0.34 to 0.11 and 0.10. Thus, the sum of singlet oxygen efficiency ΦΔ and fluorescence quantum Φf yield for these compounds is ca. 0.6 (Table S17).Triarylboranes bis‐triarylboranes sensitize 1O2 with efficiencies of 0.6, 0.8, 0.7, 0.6, and 0.6 for 1+Cat, 2+Cat, 2+Cat(i), 3+Cat, and 4+Cat, respectively , which is the reason for the raised baseline between 1230 and 1260 nm in Figures ΦΔ of ca. 0.6 is estimated for all charged bis‐triarylboranes.The charged y Figure , Table 41 excited states of 1+Cat–4+Cat thus decay ca. 40 % by fluorescence and ca. 60 % by intersystem crossing to the triplet state, which very efficiently sensitizes 1O2. Attaching two triarylboranes to a bithiophene motif leads to a more efficient energy transfer to triplet oxygen than for the mono‐triarylboranes. Thus, interactions with DNA, RNA and DNApore and cell imaging studies were only examined for the cationic bis‐triarylboranes.The Sbis‐triarylboranes 1+Cat–3+Cat and naturally occurring calf thymus DNA  and AT‐(52 %) base pairs), double stranded (ds) poly rA–poly rU  and DNApore .The stability of 1+Cat–3+Cat upon addition of increasing amounts of DNA, or RNA were studied at pH 7 and/or pH 8. In combination with the results of thermal denaturation (Tm) and circular dichroism (CD) experiments of the polynucleotides, which are known to be unique for the respective molecule, conclusions about the mode of interaction  can be drawn.[bis‐triarylboranes 2+Cat–3+Cat, suggest non‐covalent binding of the latter, characterized by the type of interactions which can increase the stability of DNA/RNA . However, the emission of 1+Cat showed negligible changes in the presence of any polynucleotide at any pH , and 3+Cat, Tm is increased compared to pure ctDNA or pApU at pH 7. Thus, these three compounds efficiently bind to ctDNA and pApU and stabilize these polynucleotides , and 3+Cat are lower than that reported for 4+Cat.In the presence of 2+Cat–3+Cat increases depending on the structure of the polynucleotide and the number of charges. Using the Scatchard equation2+Cat, 2+Cat(i), and 3+Cat bind in the minor groove of DNA or the major groove of RNA.Upon addition of ctDNA or pApU, the emission of 2+Cat, 2+Cat(i), and 3+Cat to ctDNA or pApU, a small decrease of the intensity of the CD spectra between 230 nm and 300 nm  was observed. This can be attributed to an unwinding of the double helix of the polynucleotides upon insertion of the bis‐triarylboranes. In contrast to the previously reported tetra‐cationic compound 4+Cat,2+Cat, 2+Cat(i), and 3+Cat might be non‐uniformly oriented with respect to the chiral axis of the polynucleotide, but it was not possible to derive direct structural information. Nevertheless, in analogy with our previous binding studies,[2+Cat, 2+Cat(i), and 3+Cat can be assumed.Upon addition of ‐1 Tris‐HCl, 300 mmol L−1 KCl) were increased in the presence of 2+Cat, 2+Cat(i), 3+Cat, and 4+Cat <3+Cat<4+Cat. The unusual drop of absorbance observed for 2+Cat(i) upon addition of DNA is likely due to precipitation of a 2+Cat/DNA complex upon complete unwinding.Similarly, as described above for sodium cacodylate solutions, thermal denaturation temperatures of ctDNA and DNApore in Tris‐buffer at pH 8 (15 mmol L+ Table . The den2+Cat and 2+Cat(i) upon addition of ctDNA at pH 7, at pH 8, the emission of these compounds decreases , 3+Cat, and 4+Cat decreases  and 3+Cat, a negative band at ca. 425 nm was observed, indicating a perpendicular orientation of the transition moments to the chiral axis of ctDNA at pH 8.Addition of bis‐triarylboranes studied herein, and the same experimental procedures applied, the results of these measurements can be used for comparisons within this series of compounds. For 2+Cat, weak, negative ICD bands were observed which is characteristic for a perpendicular orientation of the transition moment of the chromophore with respect to the chiral axis of the DNApore. In contrast to the CD measurements at pH 7, which did not change at wavelengths >300 nm, addition of 2+Cat(i) to DNApore at pH 8 induced strong ICD bands at 400–450 nm , 3+Cat, and 4+Cat interact with all polynucleotides investigated. Although there are some similarities, e. g. interaction with DNApore leads to emission decrease of the compounds, the number and distribution of charges affects the interaction of bis‐triarylboranes with different polynucleotides. In particular, the induced CD bands of various derivatives , 3+Cat, and 4+Cat are useful probes for further studies of the DNApore structure and interactions with novel ligands.Thus, s Figure  can be ubis‐triarylboranes to see whether they penetrate cell membranes, stain specific organelles therein, or if they have anti‐proliferative effects to A549 and WI38 cells. Confocal microscopy studies showed that the mono‐cationic compound 1+Cat does not penetrate the cells, in contrast to 2+Cat–3+Cat. Thus, this compound was not further investigated in biological studies. For 2+Cat, only preliminary results were obtained, suggesting localization at the cellular membrane and certain compartments in the cell nucleus. However, due to the low stability of this compound in Dulbecco Modified Eagle's Medium (DMEM) containing 10 % fetal bovine serum (FBS), only the staining patterns of 2+Cat(i) and 3+Cat were further investigated.Studies were conducted on the cationic 2+Cat(i) and 3+Cat was tested using the MTT assay against human lung carcinoma (A549) and normal lung (WI38) cell lines . In the dark, 2+Cat(i) is not cytotoxic in all concentrations tested . However, when irradiating the incubated cells of both cell lines in the presence of 1 μM 2+Cat(i) at 400–700 nm for 5 min, the viability of the cells was reduced drastically with increasing concentration of 2+Cat(i).The cytotoxicity of 3+Cat is not cytotoxic to both cell lines in concentrations of 0.1 μM and 1 μM. However, at 10 μM concentrations, 3+Cat is cytotoxic to A549 cells. This toxicity is even more pronounced for WI38 cells. For both cell lines, the toxicity increases under irradiation (400–700 nm) at 1 μM and 10 μM concentrations.In the dark, 2+Cat, 2+Cat(i), or 3+Cat by confocal microscopy while irradiating at 405 nm at the full power of the light source (LED). Within 2 min, significant changes in the cell morphology were observed  suggesting strong cellular damage. Simultaneously, the emission of the dyes bleached rapidly within 30 s to 1 min . The stability of the emission increases with the number of cationic charges. After the emission is quenched completely, no additional cell damage was observed. Control experiments on untreated cells did not show any sensitivity to light exposure. This is in good agreement with the cell toxicity of 2+Cat(i) and 3+Cat under UV irradiation found during the MTT assay.The increased toxicity of these compounds when irradiated with UV light results most likely from their ability to sensitize singlet oxygen (see above) as the latter is a highly reactive compound leading to cell death.2) with 2+Cat(i), or 3+Cat and exciting with light , the cells show strong green emission due to the accumulation of the compounds in some organelles in the cytoplasm. The staining patterns of both compounds were further analyzed by co‐localization experiments 2+Cat(i) (0.54) and 3+Cat (0.46), the best overlap was obtained for co‐localization with LAMP1, a staining antibody selective for lysosomes. However, the staining pattern of the bis‐triarylboranes suggests further interactions with early endosomes and golgi apparati . Thus, these new compounds are not specific for one organelle but are distributed between the organelles mentioned with some selectivity for lysosomes.However, when incubating A549 cells for 90 min , 3+Cat) and their neutral precursors 2, Neut3) are described. The compounds were isolated in good yields and the influence of different numbers and locations of charges on electrochemical and photophysical properties and cellular imaging was investigated. Photophysical investigations reveal expected behavior for D‐A compounds wherein the boron center is the electron‐acceptor in all compounds with differing electron‐donating moieties, as supported by DFT and TD‐DFT calculations. For the cationic bis‐triarylboranes, an increasing hypsochromic shift of the emission spectra with increasing dipole moment was observed in acetonitrile. CV measurements of the neutral and cationic bis‐triarylboranes show that the π‐conjugation, and therefore the communication between the boron centers, is improved upon methylation of the dimethylamino groups. Efficient singlet oxygen sensitization was demonstrated for the cationic bis‐triarylboranes 1+Cat, 2+Cat, 2+Cat(i), and 3+Cat. For comparison with the properties of the bis‐triarylboranes, selected properties were also investigated for the mono‐triarylboranes 4a‐6a, 5c, and 6c.The syntheses of selectively trimethylammonium‐substituted, bithiophene‐bridged, 1+Cat, 2+Cat, 2+Cat(i), and 3+Cat in pure water, photophysical investigations and biological studies were performed in aqueous environments containing less than 1 % acetonitrile and 0.1 % DMSO, respectively. Studies with DNA and RNA in buffered solutions show negligible binding for the mono‐cationic compound 1+Cat but strong binding for 2+Cat, 2+Cat(i), and 3+Cat within the minor groove of DNA or the major groove of RNA exhibiting a strong fluorescence increase under physiological conditions. The compounds also bind strongly to DNApore, most likely inside the pore. Thus, they could potentially act as a “stopper” of ubiquitous material flow, which DNApore enables when inserted in, e. g., bacterial membranes.2+Cat, 2+Cat(i), and 3+Cat might be useful to study the fine structure of DNApore and the interaction with other ligands. Studies with A549 and WI38 cells show all compounds to be cell permeable except for the mono‐cation 1+Cat. Under intense irradiation (400–700 nm), the emission of 2+Cat, 2+Cat(i), and 3+Cat bleaches rapidly with simultaneous changes of cell morphology, most likely due to in situ formation of singlet oxygen. Thus, the cytotoxicity of the compounds is strongly increased upon irradiation with light (400–700 nm) as demonstrated using the MTT assay. Without irradiation, 2+Cat(i) and 3+Cat are not cytotoxic up to concentrations of 10 and 1 μM, respectively, for both cell lines. Co‐localization experiments of both compounds in A549 cells demonstrate the lysosome as the main but not only accumulation site, since 2+Cat(i) and 3+Cat also localize in other organelles. Thus, the cationic bis‐triarylboranes are promising theranostic agentsbis‐triarylborane chromophores for biological applications, the number and distribution of charges should be considered as well as the bridging unit.Due to the moderate solubilities of 4a) and 2072402 (for 4b)2072401 (for  contain(s) the supplementary crystallographic data for this paper. These data are provided free of charge by the joint Cambridge Crystallographic Data Centre and Fachinformationszentrum Karlsruhe Access Structures service.”“Deposition Number(s) The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


Cerebrospinal fluid analysis contributes to the diagnosis and neuropathogenesis of neuroinvasive arboviruses. Neurological complications caused by dengue, Zika, and chikungunya infections have high clinical relevance because of their high potential to cause death or neurological deficits. We aimed to evaluate the use of cerebrospinal fluid assays for diagnostic support in neurological disorders associated with dengue, chikungunya, and Zika infections. A systematic review was carried out by searching the electronic databases LILACS, PubMed, Scopus, and Embase for articles written in English, Portuguese, or Spanish in the last 19 years. Published studies were reviewed using the terms “dengue,” “Zika”, “chikungunya”, alone or in combination with “cerebrospinal fluid” in the period from 2000 to 2019.  A total of 98,060 studies were identified; of these, 1.1%  used cerebrospinal fluid assays for neurological investigations. The most frequent neurological disorders included encephalitis (41.4%), congenital syndromes (17%), and microcephaly associated with Zika virus infections (8.9%). Neuroinvasive disorders were confirmed in 8.03% of 58,478 cases by specific cerebrospinal fluid analyses. The main methods used were IgM-specific antibodies (66%) and reverse transcription-polymerase chain reaction (10%). The largest number of scientific papers (29%) originated from Brazil, followed by India (18.4%) and the United States (14.4%). Although cerebrospinal fluid analysis is of great importance for increasing neurological diagnostic accuracy and contributes to the early diagnosis of neuroinvasive dengue, chikungunya, and Zika infections, it is underused in routine laboratory investigations worldwide.  However, CSF findings may be similar for a variety of viral agents. Therefore, accurate diagnosis depends on clinical findings and special CSF laboratory tests such as specific molecular and immunological analyses.Cerebrospinal fluid (CSF) is an ultrafiltrate of blood plasma produced by the choroid plexus cells. It protects the brain and spinal cord against trauma, provides nutrients to the nervous system tissue, and participates in the removal of residues generated by brain metabolismFlaviviridae, Togaviridae, and Bunyaviridae families. Flaviviruses  and alphaviruses (chikungunya virus) are commonly transmitted by female mosquitoes of the genus Aedes  in a vertebrate human viremic hostArboviruses are viruses that are transmitted to the vertebrate host through the bite of arthropod vectors, especially mosquitoes and ticks, and can be maintained in wild and/or urban cycles. Among the arboviruses, the main families that cause disease in humans are the ,,,,,,.Scientific literature has reported an increase in the occurrence of neuroinvasive diseases after the simultaneous circulation of the dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV) viruses. These include neurological disturbances of high morbidity and mortality such as acute encephalitis, aseptic meningitis, myelitis, and post-infection disorders such as Guillain-Barré syndrome, acute disseminated encephalomyelitis, optic neuromyelitis, cranial nerve palsy, and congenital disorders,. Therefore, CSF examination has a role in demonstrating the etiological agent in viral infections of the CNS. The presence of viral genetic material in the CSF is considered to be the strongest evidence of causality,,,,,.The diagnosis of neuroinvasive diseases associated with DENV, ZIKV, and CHIKV according to the Centers for Disease Control and Prevention (CDC) is based on the clinical criteria which include the appearance of neurological manifestations in the absence of a more likely clinical explanation, and are confirmed by laboratory criteria diagnosis, as follows: the presence of the following alone or in combination: viral RNA viral antigens in serum, CSF or other body fluids, specific IgM-reactivity in the CSF with non-reactive specific IgM reactivity for other arboviruses endemic in the region, virus-specific IgM in serum confirmed by virus-specific neutralizing antibodies, and increased titer-specific IgG in paired serumThe objective of this study was to conduct a review of the scientific literature on arboviruses  that used the CSF assay for the investigation of neurological manifestations, over a period of 19 years. The results may elucidate the magnitude of CSF-assay use in arbovirus infections and demonstrate the importance of the topic; this would favor debate and interest in the scientific community about the importance of CSF assays for diagnostic support in neurological cases resulting from DENV, ZIKV, and CHIKV infections.Published studies were reviewed using the terms “dengue”, “zika,” “chikungunya" alone and in combination with “cerebrospinal fluid” in the LILACS, PubMed, Scopus, and Embase databases. The guidelines used to report the systematic review were based on the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelinesThe articles selected for this systematic review were reviewed in detail. We analyzed the total number of publications from 2000 to 2019, the number of publications per year and country, year of publication, and publication type . We also analyzed the number of confirmed neurologic diagnoses that involved infections by DENV, ZIKV, and CHIKV that used CSF as a screening sample. From these data, we developed graphs and tables in GraphPad Prism 8.0 and/or Microsoft Office Excel 2010. We analyzed the results obtained from the databases both separately and in combination. Duplicate articles were manually deleted using the duplicate removal function of the Mendeley desktop 1.19.4 program.The selection process of the articles for this study is presented in the flowchart presented in the A total of 98,060 publications were found in the LILACS, PubMed, Scopus, and Embase databases using the single keywords “dengue,” “Zika”, and “chikungunya”. The proportion of publications using CSF for diagnostic evaluation over these 19 years was equivalent to only 1.1%  of the DENV, ZIKV, and CHIKV arbovirus articles A. For eaAccording to the countries registered by the authors and collaborators, Brazil had the largest number of scientific papers available, accounting for 302 articles, from 2000 to 2019 C. This fIn the 1,041 publications selected using the keywords “dengue”, “Zika”, and “chikungunya” both alone and in combination with “cerebrospinal fluid” over the 19 years, we identified 58,478 suspected cases of arbovirus and neurological disorders such as encephalitis, congenital syndromes, microcephaly, meningoencephalitis, meningitis, GBS, and myopathy. The CSF samples were investigated for arboviruses using different methodologies such as the detection of specific IgM and/or IgG antibodies, plate reduction neutralization test, detection of viral antigens, amplification of viral RNA by PCR techniques, and next-generation metagenomic sequencing. However, only 8.03%  were confirmed as DENV, ZIKV, and/or CHIKV cases by the analysis of CSF alone or in association with other fluids D. The diThe number of publications that evaluated arbovirus-associated neurological manifestations in combination with CSF was low , considering all four databases. Except for those pertaining to DENV , accessible works that related ZIKV  and CHIKV  to CSF during the reporting period were sparse.There was a considerable change in the number of articles published between 2016 and 2019. In the LILACS database, 17 publications were identified: six pertaining to the keywords “dengue” and “CSF,” eight to “"Zika” and “"CSF”, and three to “chikungunya” and “CSF”. In the PubMed database, 110 publications were identified: 51 pertaining to the keywords “dengue” and “CSF”, 39 to “Zika” and “"CSF”, and 20 to “chikungunya” and “CSF”. In the Scopus database, 373 publications were identified: 152 pertaining to the keywords “dengue” and “CSF”, 161 to “Zika” and “CSF”, and 60 to “chikungunya” and “CSF”. In the Embase database, 115 publications were identified: 51 pertaining to the keywords “dengue” and “CSF”, 30 to “Zika” and “CSF”, and 34 to “chikungunya” and “CSF” A. Regarding the types of publications, 17 original articles, 11 case reports, two reviews, and one cohort study were retrieved from the LILACS database. Querying the PubMed database resulted in the identification of 106 original articles, 37 case reports, five reviews, six case series, four cohort studies, four brief reports, four notes, two short communications, and two multicenter studies. The Scopus database filtered 441 original articles, 58 reviews, 58 letters, 21 notes, 10 editorials, seven conference papers, and two short surveys B. Queryi.The arboviruses DENV, ZIKV, and CHIKV are present in more than 120 subtropical and tropical countries. They emerge in poor urban areas and in the peripheral parts of the world. It is estimated that approximately 4 billion people are exposed to the risk of DENV, ZIKV, and CHIKV infectionsThis study reviewed the literature that describes the presence of DENV, ZIKV, and CHIKV in the CSF in different neurological conditions , as reported in various types of studies . The differences in the number of searches accessible among the databases were justified by the information obtained as a result of each of the searches. LILACS contains only Latin American data. The PubMed database comprises the medical libraries of the United States and 70 countries. Scopus is the world’s largest database and covers more than 5,000 international publishers. The Embase database has international coverage and contains more than 32 million records from more than 8,500 journals. This characteristic reflects its greater ability to filter scientific papers.,,,,. At this time, an increase in the number of microcephaly cases was recognized in Brazil during the ZIKV epidemic. This finding highlights the possible relationship between the neurological condition and infection with the virus. Retrospective studies also found a proliferation in other congenital brain abnormalities during the ZIKV epidemic in 2013 in French Polynesia,In 2015, no studies were identified in any of the analyzed databases when the keywords “Zika” and “CSF” were combined. The incidence of GBS associated with ZIKV infection increased in the epidemics of 2015 and 2016 in Brazil, Colombia, the Dominican Republic, El Salvador, Honduras, Suriname, and Venezuela,,,Brazil had the largest number of available scientific papers. Dengue fever, chikungunya fever, and acute disease caused by ZIKV are compulsory notifiable diseases in the country, and are included in the National List of Compulsory Notification of Diseases, Diseases, and Public Health Events, unified by Consolidation Ordinance No. 4  from the Brazilian Ministry of Health. The data recorded by the Health Surveillance Secretariat demonstrate the epidemiological relevance of these arboviruses. In 2016, 2017, 2018, and 2019, the total reported probable case numbers related to DENV in various publications , ZIKV , and CHIKV  infections in Brazil were significantIt is estimated that 0.5-21% of patients with dengue develop neurological manifestations. In 2016, a cohort study was conducted at La Réunion Island reported CHIKV infections in 42% of encephalitis casesThe etiopathogenesis of neurological manifestations may occur through the direct action of the virus, by autoimmune mechanisms, hemorrhage, and the metabolic disorders it causesOur analysis showed that from 2000 to 2019, less than 1.1% of the scientific publications in the four databases used CSF analysis to investigate arboviruses  in cases with neurological manifestations. As discussed above, considering the prevalence of neuroarboviruses and the importance of CSF for diagnosis, the number of scientific publications found in the area is underestimated. Therefore, neurological diseases caused by these arboviruses may be underdiagnosed. This is evident from our results which showed that only 8.03% of the suspected cases were diagnosed. Of these, 75% underwent CSF analysis alone or in association with other fluids. The detection of the specific IgM was the methodology that contributed the most to the diagnoses. Despite advances in the number of arbovirus investigations and the confirmation of CSF as an essential clinical sample in suspected cases with neurological manifestations, further research and more studies are needed. In addition, the authors do not exclude the possibility of a possible analysis bias in some of the studies included in this review. Among these biases, the sample size in the study, the statistical significance of the main result of a given study, and type of funding for some studies, may be included. The increase in recent years in the number of cases with a co-circulation of arboviruses  worldwide has led to a continuing need for research to clarify the pathophysiology of neurovascular diseases and complications caused by arboviruses. CSF analysis contributes to the diagnostic confirmation and elucidation of the neuropathogenesis of CNS tropical neurological diseases such as arbovirus infections. CSF analysis also plays a crucial role in excluding other possible causes of neurological disorders. In cases suspected of neurological manifestations due to viral infection, neuroarboviruses should be investigated in endemic areas. Despite the high morbidity and lethality of these arboviruses and their neurological complications, we emphasize that CSF is still rarely used for elucidation, diagnostic support, and research.


We compared the health status and ecological characteristics of farm-bred and wild frogs using veterinary clinical examinations. Wild frogs showed a better nutritional status and superior traits of locomotory performance than farm-bred frogs. However, wild animals are not necessarily in good health. This comparison can help determine the condition of animals and suggest improvements to the breeding environment.Due to the decline in the population and the difficulty of in situ conservation, several anuran species are being reared in captivity. In this study, we identified physiological and morphological differences between farm-bred and wild frogs. Nine different serum components were used as indicators of osmotic pressure, homeostatic state, organ function, and nutritional status of farm-bred frogs and wild frogs, while radiographic techniques were used to visualize differences in bone mineral density and body composition ratio. Additionally, X-ray skeletal images were used for morphological analysis to estimate differences in locomotory performance between the two groups. Wild frogs harbor traits that aid in better locomotory performance than farm-bred frogs. They also have a relatively lower fat content ratio and higher calcium and phosphorus serum levels than farm-bred frogs, suggesting a difference in nutritional status. However, hepatic stress was higher in wild frogs than in farm-bred frogs. Veterinary clinical examinations allow for the identification of differences in nutritional and morphological conditions between farm-bred and wild frogs. Determining the health of animals can help improve their living conditions, eliminate conditions that can negatively affect them, and effectively manage them on farms, in zoos, and at ex situ conservation institutes. The welfare and ethical treatment of animals are particularly important in the case of captive animals that are artificially bred for various purposes . One impBatrachochytrium dendrobatidis) are preferentially selected and bred for ex situ conservation [Several amphibian species worldwide are experiencing declining numbers and even extinction, and are consequently being bred in captivity. Therefore, identifying environmental factors that affect the health of frogs is particularly important. Some species that are sold as food or pets are raised according to local regulations or international conventions ,11,12,13ervation ,15,16. Aervation . While mervation , these aervation ,20. ThusDetermining the health status of the animals and the adequacy of their living conditions can be achieved by understanding the physical, chemical, and biological differences of habitat status between captive and wild amphibians. Captive amphibians may have a smaller range of motion, less access to food, and be exposed to a different environment than wild amphibians ,22. LongIn this study, we compared physiological and morphological parameters between farm-bred frogs and wild frogs to determine the impact of living conditions on the health status of the frogs in question. Serum chemistry and radiographic analyses were used to estimate the nutritional and physiological status of the animals. Additionally, skeletal images were analyzed to assess the range of locomotion and predatory pressure experienced by the frogs. These criteria were used as indicators of the quality of living environment and were compared between the wild frogs and the frogs bred in captivity. A comparison between males and females was also conducted to confirm whether the sex of the animal is relevant when determining its health. The examination protocol developed in this study can be adopted to monitor and improve the living conditions of frogs reared in captivity.Pelophylax nigromaculatus) were purchased from two different farms (farm-bred frogs). Farm-bred frogs were autonomously fed only crickets and mealworms, and they bred or hibernated in a greenhouse space (area of 1500–2000 m2) narrower than their wild habitats. Both farms started farming captive frogs in 2003 and 2005, respectively. Additionally, both hibernation and breeding take place within the greenhouse. Fifty black-spotted pond frogs (Pelophylax nigromaculatus) were captured from Chungcheongnam-do between 9 pm and 11 pm in May 2019 and June 2019, and again in June 2020 and July 2020 (wild frogs). Wild frogs were collected from four different rice paddy fields around reservoirs, rivers, and marshes to eliminate differences caused by local environmental variations. There were no factors that could stress the frogs, such as chemicals, noise, or light pollution around the collection sites. The appearance of the frogs was scrutinized to confirm that there were no abnormalities such as wounds, ascites, swellings, or deformities. Both farm-bred and wild frogs were confirmed to have no abnormalities in appearance after an acclimation period of 2 h. Both groups of frogs contained breeding and non-breeding individuals; however, only mature frogs with secondary sexual characteristics were used for analysis. Juvenile frogs exhibit high growth rates and could potentially contribute to large variations in the analysis; thus, they were excluded from the analysis. Males were distinguished by the presence of the nuptial pad on the front toe and the vocal sac. Adults without both characteristics were considered female. Therefore, frogs that were larger than the size of the collected males and had no male characteristics were judged to be female. Other individuals were judged as immature juvenile frogs and excluded from the analysis. In total, 35 farm-bred frogs  and 36 wild frogs  were assessed in the physiological and morphological analyses. Of the frogs not used in the analysis, 14 wild frogs were released to their original sampling sites, and 15 farm-bred frogs were bred in the Zoology Lab of Kongju National University. In total, 71 frogs (35 farm-bred and 36 wild frogs) were euthanized after blood sample collection for further analysis by spinal cord pithing, and fixed in 70% ethanol. Experimental procedures were performed in accordance with the regulations of and with the approval of the Experimental Animal Ethics Committee of Kongju National University (KNU_2019-01).From May 2019 to June 2020, 50 black-spotted pond frogs (We measured the snout-vent length (SVL) in the order of 0.01 mm units using a digital caliper. This measurement is an indicator of amphibian body size. Body weights were also measured using a digital balance in the order of 0.01 g units. These measurements were used to confirm the difference in basic physical condition between farm-bred and wild frogs as well as between female and male frogs.g, 10 min) to obtain serum. All samples were stored at −40 °C until further analysis.Chemical anesthesia can affect the muscle physiology in frogs . TherefoA clinical chemistry automated analyzer  was used to analyze nine serum components, namely total protein (TP), albumin, glucose, calcium, phosphorus, alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) , and creDual energy X-ray absorptiometry  was used to measure body composition and BMD. DEXA helps calculate bone mineral content (BMC), fat content, and lean body content with the use of alternately transmitting high-and low-energy X-rays a. Of thep-value of this distance. We also used a graph that matched the average skeletal shape of the four groups with an average skeletal shape of each group, and compared the morphological distance.We used landmark-based geometric morphometrics to analyze skeletal shape. TpsDig software  was usedp < 0.05.A two-way ANOVA test was used to compare the difference of each content  between farm-bred and wild frogs, and between male and female frogs. Two-way ANOVA was employed to identify interaction effects between the two main factors (Group*Sex). If interaction effects were present, Tukey’s post hoc test were performed on each group . Statistical analyses were performed by GraphPad Prism version 7.0 for Windows . Morphological differences in skeletal shape between females and males and between farm-bred and wild frogs were analyzed by CVA in Morpho J. All statistical differences were considered to be significant at p > 0.05) between the two main groups (Group*Sex). The frogs did not significantly differ (p > 0.05) in SVL (F = 0.111) or body weight (F = 5.161 × 10−6) between the farm and wild groups. However, SVL (F = 4.161) and body weight (F = 9.279) of female frogs were significantly higher (p < 0.05) than in male frogs (Neither SVL (F = 0.819) nor body weight (F = 0.296) were significantly different  between groups (farm-wild) or between sexes.Glucose, BUN, and creatinine did not significantly differ (p < 0.05) than that of farm-bred frogs, but this was not the case between sexes (p > 0.05). The wild frogs had significantly higher levels (p < 0.05) of ALT than farm-bred frogs, and the female frogs had significantly higher levels (p < 0.05) of ALT than male frogs.AST of wild frogs was significantly higher (p < 0.05) among the four groups, whereas the ALTs of the other three groups did not significantly differ (p > 0.05).Notably, the ALT of wild female frogs had the highest significance (p > 0.05) between farm-bred frogs and wild frogs, whereas the female frogs had significantly higher (p < 0.05) TP and albumin levels than the male frogs.TP and albumin values were not significantly different (p < 0.05) serum levels of calcium and phosphorus than the farm-bred frogs. The female frogs had significantly higher (p < 0.05) calcium levels than male frogs, whereas phosphorus did not significantly differ (p > 0.05) between the sexes.Wild frogs had significantly higher (p > 0.05) in the two main factors (Group*Sex). Wild frogs had a significantly higher (p < 0.05) BMC (F = 4.485) and lean body contents (F = 6.675) ratios, and a lower fat contents ratio (F = 7.347) than farm-bred frogs, but there was no significant difference in BMD  between the two groups (p > 0.05) between female and male frogs.BMC (F = 0.293), fat contents (F = 1.063), lean body contents (F = 0.756) ratios, and BMD (F = 0.028) did not have a significant interaction effect . In the skull shape analysis, CV1 accounted for 57.03% of morphological variance, and individuals with a higher CV1 represented a longer and narrower skull shape. CV2 accounted for 25.03% of morphological variance, and the individuals with a higher CV2 represented a shorter and wider skull shape a. Farm-bIn the skeletal analysis of the lower body, CV1 accounted for 61.47% of morphological variance, where the individuals with a higher CV1 represented a wider pelvis with shorter ilium and urostyle. CV2 accounted for 26.05% of morphological variance, and the individuals with a higher CV2 represented a longer and narrower pelvis with shorter urostyle b. The frWe confirmed the significance of morphological differences among four groups by calculating Mahalanobis distance in CVA . In the Likewise, there were differences between farm-bred and wild frogs in the morphological analysis of skeletal shape of the lower body. However, there was no significant difference between female and male wild frogs. Contrarily, the farm-bred female frogs and farm-bred male frogs showed a significant difference in lower body shape. Wild female frogs had a longer pelvis with a longer urostyle. Similarly, the wild male frogs had a narrower pelvis with longer urostyle. Farm-bred female frogs had a similar lower body shape to the average shape of the four groups, and farm-bred male frogs had a wider pelvis with shorter urostyle.In this study, nutritional, physiological, and morphological differences were compared between farm-bred and wild frogs. Some serum components differed between male and female frogs. AST and ALT, indicators of hepatic function, were higher in wild frogs than in farm-bred frogs. Calcium and the phosphorus levels in wild frogs were higher than those in farm-bred frogs, suggesting differences in the composition of diet and nutrients consumed by both groups. Body composition did not differ between males and females, while the farmed-bred frogs had a higher fat contents ratio and a lower lean body contents ratio than wild frogs. Although skull shape was not different between males and females, there was a significant difference between wild frogs and farm-bred frogs. Similarly, the skeletal shape of the lower body of wild frogs was significantly different from farm-bred frogs. In the farm-bred frogs, lower body shape was also significantly different between farm-bred female frogs and male frogs. These differences indicate that the body shape of wild frogs suggests a better jumping performance than the body shape of farm-bred frogs.Wild frogs generally require better locomotory performance than farm-bred frogs ,44. ThisThe fat content of farm-bred frogs was higher than that of wild frogs. This may be due to differences in locomotory performance or food consumed between farm-bred and wild frogs. Differences in the locomotory performance of frogs can change their energy requirements , and conContrary to nutritional status, AST and ALT levels, which are indicators of the hepatic state, were higher in wild frogs. Wild animals are exposed to several stressors, such as physical, chemical, and biological stresses. Examples include traffic noise, exposure to salinity, and predation. Exposure to these triggers the release of corticosterone, a stress hormone in amphibians ,53,54. HVarious types of veterinary examination can help improve the inadequate captive environment by making it possible to predict the exact condition of the individual. However, some serum components  showed differences between males and females. This corroborates previous studies ,63 and sWe confirmed how the physiological and morphological adaptive responses in farm-bred frogs differ from those in wild frogs. Morphological analysis and physiological analysis helped us to predict how the nutritional and spatial conditions of farm frogs and wild frogs differ. Although some health-related components seemed to be better in wild frogs, the health conditions of frogs from wild populations were not unconditionally better than those of farm-bred frogs. Understanding the health condition of frogs and improving the environment that may negatively affect them can aid in promoting more efficient animal management and environmental enrichment. These examinations will be suitable for use on farms and in zoos, as well as in ex situ conservation institutions to diagnose the health status of individuals and to secure healthy animals.


Anti-human leukocyte antigen antibodies (anti-HLA) play a crucial role in graft. Detection of anti-HLA, both pre- and post-transplant is a crucial investigation in clinical organ transplantation.Three methodologies for the detection of lymphocytotoxic antibodies were compared to establish which of these is best suited to optimise pre-transplant donor-recipient matching.Serum samples from 15 renal transplant patients were tested for the presence of anti-HLA by i) cytotoxic-dependent cross-match (CDCXM), ii) flow cytometric cross-match (FCXM) and iii) Luminex-based donor specific antibody cross-match (DSAXM) method, Confirmatory tests for the presence of preformed HLA antibodies were tested using Luminex methodology.Two (13%) of the 15 patients had positive HLA Class I antibodies (Ab) using all 3 methods. An additional 2 HLA Class I Ab were identified with FCXM/CDCXM. DSAXM identified 1 HLA Class I positive, not indicated by CDCXM/FCXM.High HLA Class II positivity (40%), identified by CDCXM, while DSAXM and FCXM identified two and one patients, respectively. CDCXM produced 4 false-positive results confirmed by lymphocyte single antigen (LSA) assay.The DSAXM method appears to add value in pre-transplantation screening to identify pre-sensitised patients that may not reject the donor graft due to the absence of donor-specific antibodies. Chronic kidney disease (CKD) is a worldwide public health problem. In this context, renal replacement therapy can be done either by dialysis or organ transplantation. However, dialysis is life-long and is associated with reduced quality of life and increased risk of mortality. Kidney transplantation, on the other hand, offers better survival and quality of life benefit for patients with endstage kidney disease relative to dialysisAccording to the South African Renal Registry Annual Report of 2015, the total number of patients on renal replacement therapies (RRT) was 10 360. Gauteng province had the highest number of patients at 3238 (958 from public sector and 2280 from the private sector). Of the 10 360 patients, 1 440 13.9%) were on peritoneal dialysis and 7 529 (72.2%) were on haemodialysis. Of these patients, only 1 391 (13.4%) underwent renal transplantation.9% were It is imperative therefore, that available organs are optimally utilised by ensuring that best-practice methods are applied when screening for potential rejection risk. In this context, the impact of detection of pre-formed human leukocyte antigen (HLA) antibodies reactive with transplanted organs is a well-established practice in clinical renal transplantationPresently, complement dependent cytotoxicity cross-match (CDCXM) remains the most frequently utilised pre-transplant cross-match technique in the South African settingst for renal transplantation at the Steve Biko Academic State Hospital and Jacaranda Private Hospital, Pretoria, South Africa, were enrolled in this study, which was conducted from September 2014 through April 2015.Fifteen patients and their living – related donors, who were candidates undergoing their 1Unfortunately, there is no known details collected at the start of the study regarding potential multiparous or previous transfusions. Written and signed informed consent was obtained from each patient and healthy donor prior to enrolment into the study. The study was approved by the Research Ethics Committee of the Faculty of Health Sciences of the University of Pretoria, South Africa and conformed to good laboratory practice, as well as with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Ethics certificate reference number: 242/2013.The patients were cross-matched with their potential donors using three different methods viz the Terasaki microlymphocytotoxicity technique (CDCXM), the flow cytometry cross-match (FCXM) and the Luminex-based donor specific antibodies cross-match (DSAXM).CDCXM: This assay was performed using the standard two-stage National Institutes of Health (NIH) technique . Donor T- and B-lymphocytes were isolated via attachment to CD2- and CD19-monoclonal antibody-bound immunomagnetic beads respectively, followed by elution .Briefly, donor T- and B- lymphocytes were incubated with recipient sera for 30 min at room temperature. Rabbit complement was added to each well and the microplates incubated for 60 min at room temperature. The cells were then stained with a fluorescent dye mix, containing acridine orange (AO) and ethidium bromide (EB)  and observed microscopically for cytotoxicity using a fluorescence microscope. DSA (donor specific antigen) binds to the donor cells, activating the complement cascade via the classical pathway, resulting in lysis of lymphocytes detected by differential fluorescence . CDCXM results for both T- and B-cells were considered positive when cell death exceeded that of the negative control by 20%.FCXM: This assay was performed using patients' serum samples and donor peripheral blood mononuclear cells. The technique was performed by mixing 100µl of a 1x106 cells/ml donor lymphocytes with 20µl of patient or control sera and incubated for 60 minutes at room temperature, followed by three washes with phosphate-buffered saline . To identify alloantibodies and differentiate between T and B cells, the re-suspended pellet was then incubated for 30 min in the dark with 5µl of phycoerithrin-CY5 labelled anti-CD3 (CD3-PC5) , 5µl of phycoerithrin labelled anti-CD19 (CD19-PE) monoclonal antibody , and 10µl of goat F(ab)2 antihuman IgG - fluorescein isothiocynate (FITC) antibody . Two wash steps were then performed as described above and cells analysed following re-suspension in 200µl of PBS.Multicolour flow cytometric analysis was performed using the Cytomics FC 500 instrument . Viable lymphocytes were gated on the basis of their forward and side- scatter characteristics. Flow cytometry crossmatch results were expressed as the median channel florescence shift from the negative serum of either the T- and/or the B-cells.DSAXM: This assay was performed as per the manufacturer's instructions . It is used to detect IgG antibodies to donor-specific class I and class II HLA. Donor lymphocytes were isolated from peripheral blood, solubilised with non-ionic detergents, followed by centrifugation to remove cell debris and the supernatant (lymphocyte lysate) decanted.Donor lysates were then incubated with capture beads at room temperature for 30 min in the dark with intermittent mixing to facilitate binding of the solubilized donor HLA molecules to the immunofluorescent beads. The assay included control beads to monitor the amount of background fluorescence in the assay.After incubation, beads/lysate mixtures were transferred to the filter plate and washed. Diluted recipient serum was then added to the beads and incubated with agitation in the dark for 30 min followed by 3 washes. Diluted anti-human IgG streptavidin-phycoerythrin conjugate (SAPE) was then added and incubated for a further 30 min period. After addition of wash buffer, readings were taken on the Bio-Plex array system, Luminex 100 analyser.Determination if a captured bead was positive or negative for DSAXM was performed according to the manufacturer's instructions. The mean fluorescence intensity (MFI) value of each bead was compared to three cut-off values . The cut-off values were calculated from the measured background of three negative control beads in each test well. The process was repeated for each of the two beads to obtain three results . A sample was considered positive if two or more Adjusted MFI Values were positive and a sample was considered to be negative if two or more Adjusted MFI Values were negativeHLA antibody analysis: In this assay, serum samples from the recipients were analysed for HLA class I and class II IgG HLA antibodies using the commercially available LABScreen mix, single antigen (LSA) class I and class II assay kit  on a Luminex platform . The procedure was performed according to the manufacturer's instructions. Briefly, serum was first incubated with LABScreen® beads coated with HLA antigens , using a 96 V-shaped well plate, followed by washing to remove unbound antibodies. Alloantibodies present in the test serum bind to the antigen-coated beads and are detected by addition of R-Phycoerythrin (PE)-conjugated goat anti-human IgG in the second incubation step. All incubations were performed on a gently rotating platform in the dark at room temperature. After the last wash, buffer was added to each well and samples analysed. Trimmed Mean Fluorescence values were recorded using Luminex 100 IS v 2.3 software  for data analysis. Negative and positive controls were included with each assay. Data was normalised to the background value of the negative control serum and a minimum of 50 beads was required for the assay to pass. Data analysis was performed using HLA Fusion 3.0 software . To determine the presence of HLA antibodies in the patients' sera, Mean Fluorescence Intensity (MFI) of the bead was normalized against the MFI of the negative control serum. A bead was regarded as being positive if the MFI value was 1000 or higher. This procedure enables detection of the entire spectrum of HLA allo-antibodies in recipient serum.The recipient group consisted of 9 (60%) females and 6 (40%) males, while the living related donor group consisted of 6 (40%) females and 9 (60%) males.The mean age of the recipients was 39.3 (range 21–61 years) with Caucasians accounting for 73%. Of these, the mean donor age was 41.7 (range 23–58 years) with Africans accounting for 27% of this group. Recipients and their potential donors were all blood group compatible. Donor and recipient profiles are shown in As shown in CDCXM, FCXM and DSAXM results performed on all recipients included in this study are presented collectively in Renal transplantation is the option of choice in RRT for patients with end-stage kidney diseaseThe presence of these antibodies, especially DSA, plays a crucial role in graft rejection and eventually graft lossLuminex DSAXM is a novel procedure in our local setting. To our knowledge, this procedure is not widely used for routine or diagnostic purposes in tissue immunology centres in South Africa, making our study novel in this regard.Although DSAXM Class II false results have occasionally been reportedThe DSA and FCXM methods showed equivalent performance. However, one patient with a positive FCXM class II reaction was negative with DSAXM. It was established that this patient was pre-sensitised, as the presence of HLA class II antibodies were detected as per LSA as shown in The main limitation of this study was the small numbers of subjects recruited overall, particularly for the post-transplant study. One of the reasons being that most recipients were not residing in Gauteng in close proximity to the hospitals.Hence, they were unavailable for follow-up study. Overall, the time period for this study was also limited being a post-graduate study set within a timeframe of 2–3 years and the fact that the Greater Tshwane transplantation institutions do limited LRD transplantations. Nevertheless, potentially interesting findings with translational potential resulted from the study.There have been reports by others of false-positive DSAXM Class II results