Neuronal hypofunction and network dysfunction in a mouse model at an early stage of tauopathy.

We previously reported altered neuronal Ca 2+ dynamics in the motor cortex of 12-month-old JNPL3 tauopathy mice during quiet wakefulness or forced running, with a tau antibody treatment significantly restoring the neuronal Ca 2+ activity profile and decreasing pathological tau in these mice 1 . Whether neuronal functional deficits occur at an early stage of tauopathy and if tau antibody treatment is effective in younger tauopathy mice needed further investigation. In addition, neuronal network activity and neuronal firing patterns have not been well studied in behaving tauopathy models. In this study, we first performed in vivo two-photon Ca 2+ imaging in JNPL3 mice in their early stage of tauopathy at 6 months of age, compared to 12 month old mice and age-matched wild-type controls to evaluate neuronal functional deficits. At the animal level, frequency of neuronal Ca 2+ transients decreased only in 6 month old tauopathy mice compared to controls, and only when animals were running on a treadmill. The amplitude of neuronal transients decreased in tauopathy mice compared to controls under resting and running conditions in both age groups. Total neuronal activity decreased only in 6 month old tauopathy mice compared to controls under resting and running conditions. Within either tauopathy or wild-type group, only total activity decreased in older wild-type animals. The tauopathy mice at different ages did not differ in neuronal Ca 2+ transient frequency, amplitude or total activity. In summary, neuronal function did significantly attenuate at an early age in tauopathy mice compared to controls but interestingly did not deteriorate between 6 and 12 months of age. A more detailed populational analysis of the pattern of Ca 2+ activity at the neuronal level in the 6 month old cohort confirmed neuronal hypoactivity in layer 2/3 of primary motor cortex, compared to wild-type controls, when animals were either resting or running on a treadmill. Despite reduced activity, neuronal Ca 2+ profiles exhibited enhanced synchrony and dysregulated responses to running stimulus. Further ex vivo electrophysiological recordings revealed reduction of spontaneous excitatory synaptic transmission onto and in pyramidal neurons and enhanced excitability of inhibitory neurons in motor cortex, which were likely responsible for altered neuronal network activity in this region. Lastly, tau antibody treatment reduced pathological tau and gliosis partially restored the neuronal Ca 2+ activity deficits but failed to rescue altered network changes. Taken together, substantial neuronal and network dysfunction occurred in the early stage of tauopathy that was partially alleviated with acute tau antibody treatment, which highlights the importance of functional assessment when evaluating the therapeutic potential of tau antibodies. Highlights: Layer 2/3 motor cortical neurons exhibited hypofunction in awake and behaving mice at the early stage of tauopathy.Altered neuronal network activity disrupted local circuitry engagement in tauopathy mice during treadmill running.Layer 2/3 motor cortical neurons in tauopathy mice exhibited enhanced neuronal excitability and altered excitatory synaptic transmissions.Acute tau antibody treatment reduced pathological tau and gliosis, and partially restored neuronal hypofunction profiles but not network dysfunction.

[Genetic characterization and drug resistance analysis of Salmonella Kentucky ST314 in Shenzhen in 2010-2021].

OBJECTIVE: To understand the prevalence, genetic characteristics and drug resistance features of Salmonella Kentucky ST314 in Shenzhen. METHODS: Whole genome sequencing of 14 strains of Salmonella Kentucky ST314 collected from 2010-2021 by the Foodborne Disease Surveillance Network of Shenzhen Center for Disease Control and Prevention for phylogenetic evolutionary analysis, drug resistance gene and plasmid detection; drug susceptibility experiments were performed by micro-broth dilution method. RESULTS: A total of 57 strains of Salmonella Kentucky were collected from the foodborne disease surveillance network, 14 of which were ST314. The Shenzhen isolates were clustered with isolates from Southeast Asian countries such as Vietnam and Thailand on clade 314.2, and the single nucleotide polymorphism distance between local strains in Shenzhen was large, indicating dissemination. In this study, a total of 17 drug resistance genes/mutations in 9 categories were detected in the genome of Salmonella Kentucky ST314, carrying 3 extended spectrum beta-lactamases(ESBLs), including bla_(CTX-M-24)(14.3%, 2/14), bla_(CTX-M-55)(7.1%, 1/14), and bla_(CTX-M-130)(14.3%, 2/14), all located on plasmids. Regarding quinolone resistance factors, two plasmid-mediated quinolone resistance(PMQR) genes were identified in the genome: qnrB6(71.4%, 10/14) and aac(6')Ib-cr(78.6%, 11/14), a quinolone resistance quinolone resistance-determining regions(QRDR) mutation T57 S(100%, 14/14). The multi-drug resistance rate of Salmonella Kentucky ST314 in Shenzhen was 92.86%(13/14)with the highest rate of resistance to tetracycline and cotrimoxazole(100%, 14/14), followed by chloramphenicol(92.86%, 13/14), cefotaxime and ampicillin(78.57%, 11/14), ciprofloxacin and nalidixic acid(71.43%, 10/14), and ampicillin-sulbactam had the lowest resistance rate(21.43%, 3/14). CONCLUSION: ST314 is the second most prevalent ST type among Salmonella Kentucky in Shenzhen, mainly isolated from food, especially poultry; phylogenetic analysis suggests that ST314 is a disseminated infection and the genome shows a highly genetically conserved phenotype. Drug resistance of Salmonella Kentucky ST314 is very serious, especially QRDR mutation, PMQR gene co-mediated quinolone resistance and plasmid-mediated cephalosporin resistance are prominent and deserve extensive attention.

Single-Domain Antibody-Based Protein Degrader for Synucleinopathies.

Synucleinopathies are a group of neurodegenerative diseases characterized by the accumulation of α-synuclein (α-syn) in the brain, leading to motor and neuropsychiatric symptoms. Currently, there are no known cures for synucleinopathies, and treatments mainly focus on symptom management. In this study, we developed a single-domain antibody (sdAb)-based protein degrader with features designed to enhance proteasomal degradation of α-syn. This sdAb derivative targets both α-syn and Cereblon (CRBN), a substrate-receptor for the E3-ubiquitin ligase CRL4CRBN, and thereby induces α-syn ubiquitination and proteasomal degradation. Our results indicate that this therapeutic candidate enhances proteasomal degradation of α-syn, in addition to the endogenous lysosomal degradation machinery. By promoting proteasomal degradation of α-syn, we improved clearance of α-syn in primary culture and mouse models of synucleinopathy. These findings indicate that our sdAb-based protein degrader is a promising therapeutic candidate for synucleinopathies. Considering that only a small percentage of antibodies enter the brain, more potent sdAbs with greater brain entry than whole antibodies could enhance clinical benefits of antibody-based therapies.

Pathogenetic detection, retrospective and pathogenicity analysis of a fatal case of Vibrio vulnificus in Shenzhen, China.

We report a 36-year-old male patient died of V. vulnificus-induced septicaemia and multiple organ failure syndrome after oyster consumption at a restaurant. We isolated and identified V. vulnificus vv16015 from the patient's blood sample and antibiotic susceptibility tests indicated sensitivity to all 21 antibiotics. Oyster samples were subsequently collected from the restaurant's supplier and three strains of V. vulnificus were isolated. Whole genome sequencing and analysis revealed vv16015 to be distantly related to these strains and confirmed that V. vulnificus contamination was present in the seafood of the restaurant and supplier. Using a Galleria mellonella larvae infection model, the virulence of vv16015 was determined to be higher than that of comparison strains isolated from a surviving patient (vv15018) and an oyster (vv220015). The human and environment distribution of V. vulnificus in Shenzhen is sporadic and heterogeneous, and vv16015 is highly virulent compared to other strains.

Tau-targeting therapies for Alzheimer disease: current status and future directions.

Alzheimer disease (AD) is the most common cause of dementia in older individuals. AD is characterized pathologically by amyloid-ß (Aß) plaques and tau neurofibrillary tangles in the brain, with associated loss of synapses and neurons, which eventually results in dementia. Many of the early attempts to develop treatments for AD focused on Aß, but a lack of efficacy of these treatments in terms of slowing disease progression led to a change of strategy towards targeting of tau pathology. Given that tau shows a stronger correlation with symptom severity than does Aß, targeting of tau is more likely to be efficacious once cognitive decline begins. Anti-tau therapies initially focused on post-translational modifications, inhibition of tau aggregation and stabilization of microtubules. However, trials of many potential drugs were discontinued because of toxicity and/or lack of efficacy. Currently, the majority of tau-targeting agents in clinical trials are immunotherapies. In this Review, we provide an update on the results from the initial immunotherapy trials and an overview of new therapeutic candidates that are in clinical development, as well as considering future directions for tau-targeting therapies.

Emergence of chromosomally located blaCTX-M-14b and qnrS1 in Salmonella enterica serotype Kentucky ST198 in China.

Highly fluoroquinolone-resistant Salmonella enterica serotype Kentucky has become widespread in recent years, largely associated with the spread of sequence type 198 (ST198), which often leads to multidrug resistance. Research on the genomic epidemiology of Salmonella Kentucky in China is currently uncommon. In this study, we analysed the genomic epidemiology and antimicrobial resistance characteristics of Salmonella Kentucky ST198 collected from foodborne disease surveillance in Shenzhen, China, during 2010-2021, using whole-genome sequencing and antibiotic susceptibility testing. In addition, 158 global Salmonella Kentucky ST198 genomes were included for comparison. Among 8559 Salmonella isolates, 43 Salmonella Kentucky ST198 isolates were detected during 2010-2021. The global Salmonella Kentucky ST198 evolutionary tree was divided into five clades, with Shenzhen isolates distributed in clades 198.1, 198.2-1 and 198.2-2, mainly clustered with Chinese strains. Strains in clade 198.2 dominated in Shenzhen and all of them showed multidrug resistance. Nine strains showed high resistance to ceftriaxone, which was associated with blaCTX-M-14b in clade 198.2-1, which was demonstrated to be located on the chromosome. Fifteen strains showed high resistance to ciprofloxacin, which was associated with carriage of qnrS1 in clade 198.2-2. qnrS1 was first located on an IncHI2 plasmid and then transferred into the chromosome. Here we report the genomic and antimicrobial resistance characterisation of Salmonella Kentucky ST198 in Shenzhen. Of particular concern, we identified for the first time a clade 198.2-1 isolate carrying blaCTX-M-14b as well as chromosomally located qnrS1 in clade 198.2-2 of Salmonella Kentucky ST198 in China, highlighting the necessity of surveillance of clade 198.2.

Single-domain antibody-based noninvasive in vivo imaging of α-synuclein or tau pathology.

Intracellular deposition of α-synuclein and tau are hallmarks of synucleinopathies and tauopathies, respectively. Recently, several dye-based imaging probes with selectivity for tau aggregates have been developed, but suitable imaging biomarkers for synucleinopathies are still unavailable. Detection of both of these aggregates early in the disease process may allow for prophylactic therapies before functional impairments have manifested, highlighting the importance of developing specific imaging probes for these lesions. In contrast to the ß sheet dyes, single-domain antibodies, found in camelids and a few other species, are highly specific, and their small size allows better brain entry and distribution than whole antibodies. Here, we have developed such imaging ligands via phage display libraries derived from llamas immunized with α-synuclein and tau preparations, respectively. These probes allow noninvasive and specific in vivo imaging of α-synuclein versus tau pathology in mice, with the brain signal correlating strongly with lesion burden. These small antibody derivatives have great potential for in vivo diagnosis of these diseases.

Single domain antibodies targeting pathological tau protein: Influence of four IgG subclasses on efficacy and toxicity.

BACKGROUND: Eleven tau immunoglobulin G (IgG) antibodies have entered clinical trials to treat tauopathies, including Alzheimer's disease, but it is unclear which IgG subclass/subtype has the ideal efficacy and safety profile. Only two subtypes, with or without effector function, have been examined in the clinic and not for the same tau antibody. The few preclinical studies on this topic have only compared two subtypes of one antibody each and have yielded conflicting results. METHODS: We selected two single domain antibodies (sdAbs) derived from a llama immunized with tau proteins and utilized them to generate an array of Fc-(sdAb)2 subclasses containing identical tau binding domains but differing Fc region. Unmodified sdAbs and their IgG subclasses were tested for efficacy in primary cultures and in vivo microdialysis using JNPL3 tauopathy mice. FINDINGS: Unmodified sdAbs were non-toxic, blocked tau toxicity and promoted tau clearance. However, the efficacy/safety profile of their Fc-(sdAb)2 subclasses varied greatly within and between sdAbs. For one of them, all its subtypes were non-toxic, only those with effector function cleared tau, and were more effective in vivo than unmodified sdAb. For the other sdAb, all its subtypes were toxic in tauopathy cultures but not in wild-type cells, suggesting that bivalent binding of its tau epitope stabilizes a toxic conformation of tau, with major implications for tau pathogenesis. Likewise, its subclasses were less effective than the unmodified sdAb in clearing tau in vivo. INTERPRETATION: These findings indicate that tau antibodies with effector function are safe and better at clearing pathological tau than effectorless antibodies, Furthermore, tau antibodies can provide a valuable insight into tau pathogenesis, and some may aggravate it. FUNDING: Funding for these studies was provided by the National Institute of Health (R01 AG032611, R01 NS077239, RF1 NS120488, R21 AG 069475, R21 AG 058282, T32AG052909), and the NYU Alzheimer's Disease Center Pilot Grant Program (via P30 AG008051).

Outbreak dynamics of foodborne pathogen Vibrio parahaemolyticus over a seventeen year period implies hidden reservoirs.

Controlling foodborne diseases requires robust outbreak detection and a comprehensive understanding of outbreak dynamics. Here, by integrating large-scale phylogenomic analysis of 3,642 isolates and epidemiological data, we performed 'data-driven' outbreak detection and described the long-term outbreak dynamics of the leading seafood-associated pathogen, Vibrio parahaemolyticus, in Shenzhen, China, over a 17-year period. Contradictory to the widely accepted notion that sporadic patients and independent point-source outbreaks dominated foodborne infections, we found that 71% of isolates from patients grouped into within-1-month clusters that differed by ≤6 single nucleotide polymorphisms, indicating putative outbreaks. Furthermore, we showed that despite the long time spans between clusters, 70% of them were genomically closely related and were inferred to arise from a small number of common sources, which provides evidence that hidden persistent reservoirs generated most of the outbreaks rather than independent point-sources. Phylogeographical analysis further revealed the geographical heterogeneity of outbreaks and identified a coastal district as the potential hotspot of outbreaks and as the hub and major source of cross-district spread events. Our findings provide a comprehensive picture of the long-term spatiotemporal dynamics of foodborne outbreaks and present a different perspective on the major source of foodborne infections, which will inform the design of future disease control strategies.

Investigation and Identification of Food Poisoning Caused by Clostridium botulinum Type B1 in Shenzhen, China.

Clostridium botulinum produces botulinum neurotoxins (BoNTs), which cause people who ingest them to become seriously ill and sometimes die. In recent years, sporadic food poisoning cases associated with C. botulinum have occurred across the world. In 2016, two men were admitted to our hospital in Shenzhen, China, with foodborne botulism. In this study, we report on these two typical C. botulinum-related food poisoning incidents and the steps taken to identify and characterize the causative pathogen. We characterized the bacterial pathogen isolated from the first patient using cooked meat medium and egg yolk agar bacterial cultures under anaerobic conditions, and morphologically identified the isolate using Gram staining. The in vivo bioassay results in mice showed that the minimum lethal dose of the BoNTs produced by our isolate was 0.001-0.0001 mg/mL (LD50 of the culture was estimated to be 1.5812 mg/kg). Whole genome sequencing (WGS) results showed that the isolate was identified as C. botulinum B1 Okra. The causative strain was successfully isolated from the intestinal lavage fluid collected from the initial patient.

Population dynamics and antimicrobial resistance of Salmonella Derby ST40 from Shenzhen, China.

Salmonella enterica subsp. enterica serovar Derby (S. Derby) is one of the most common serotypes responsible for salmonellosis in humans and animals. The two main sequence types (ST) observed in China are ST40 and ST71, with ST40 presently being the most common in Shenzhen. Recent years have seen an increasing number of cases of salmonella caused by ST40 S. Derby, but the epidemiology is not clear. We gathered 314 ST40 S. Derby isolates from food and patient samples for 11 years in Shenzhen; 76 globally prevalent representative strains were also collected. Whole-genome sequencing (WGS) combined with drug resistance phenotyping was used to examine population structural changes, inter-host associations, drug resistance characteristics, and the food-transmission risks of ST40 S. Derby in Shenzhen over this period. The S. enterica evolutionary tree is divided into five clades, and the strains isolated in Shenzhen were primarily concentrated in Clades 2, 4, and 5, and thus more closely related to strains from Asian (Thailand and Vietnam) than European countries. Our 11-year surveillance of S. Derby in Shenzhen showed that Clades 2, 4, and 5 are now the dominant epidemic branches, and branches 2 and 5 are heavily multi-drug resistant. The main resistance pattern is ampicillin-tetracycline-ciprofloxacin-chloramphenicol-nalidixic acid-streptomycin-sulfamethoxazole/trimethoprim. This may lead to a trend of increasing resistance to ST40 S. Derby in Shenzhen. Using a segmentation of ≤3 SNP among clone clusters, we discovered that Clades 2 and 4 contained multiple clonal clusters of both human- and food-derived strains. The food-derived strains were mainly isolated from pig liver, suggesting this food has a high risk of causing disease outbreaks in Shenzhen.

The utility of diffusion-weighted imaging and ADC values in the characterization of mumps orchitis and seminoma.

BACKGROUND: Diffusion-weighted imaging (DWI) can quantitatively reflect the diffusion characteristics of tissues, providing a theoretical basis for qualitative diagnosis and quantitative analysis of a disease. PURPOSE: To characterize testicular lesions that present as a hypointense signal on magnetic resonance imaging (MRI) T2-weighted images using DWI. MATERIAL AND METHODS: Study participants were divided into three groups. Group A were healthy controls (n = 35), group B included patients with mumps orchitis (n = 20), and group C included patients with seminoma (n = 15). DWI sequences used b-values of 0, 1000, and 2000 s/mm2. Apparent diffusion coefficient (ADC) values between 1000 and 2000 s/mm2 were calculated by MRI postprocessing software. The Kruskal-Wallis test and receiver operating characteristic analysis were performed to evaluate how well ADC values distinguished between mumps orchitis and seminoma. RESULTS: Normal testicular tissue showed a hyperintense signal on DWI and hypointensity on the ADC map: mean ADC value was 0.77 (0.69-0.85) ± 0.08 ×10-3 mm2/s. Mumps orchitis and seminoma showed slight hyperintensity on DWI: mean ADC values were 0.85 (0.71-0.99) ± 0.15 ×10-3 mm2/s and 0.43 (0.39-0.47) ± 0.04 × 10-3 mm2/s, respectively. There were statistically significant differences in mean ADC values between normal testicular tissue and seminoma and between mumps orchitis and seminoma. The cutoff ADC value for differentiating seminoma from mumps orchitis was 0.54 × 10-3 mm2/s. The sensitivity, specificity, and Youden Index for diagnosing seminoma were 99%, 31%, and 30%, respectively. CONCLUSION: High b-value DWI has potential utility for differentiating mumps orchitis from seminoma in the clinical setting.

Targeting tau only extracellularly is likely to be less efficacious than targeting it both intra- and extracellularly.

Aggregation of the tau protein is thought to be responsible for the neurodegeneration and subsequent functional impairments in diseases that are collectively named tauopathies. Alzheimer's disease is the most common tauopathy, but the group consists of over 20 different diseases, many of which have tau pathology as their primary feature. The development of tau therapies has mainly focused on preventing the formation of and/or clearing these aggregates. Of these, immunotherapies that aim to either elicit endogenous tau antibodies or deliver exogenous ones are the most common approach in clinical trials. While their mechanism of action can involve several pathways, both extra- and intracellular, pharmaceutical companies have primarily focused on antibody-mediated clearance of extracellular tau. As we have pointed out over the years, this is rather surprising because it is well known that most of pathological tau protein is found intracellularly. It has been repeatedly shown by several groups over the past decades that antibodies can enter neurons and that their cellular uptake can be enhanced by various means, particularly by altering their charge. Here, we will briefly describe the potential extra- and intracellular mechanisms involved in antibody-mediated clearance of tau pathology, discuss these in the context of recent failures of some of the tau antibody trials, and finally provide a brief overview of how the intracellular efficacy of tau antibodies can potentially be further improved by certain modifications that aim to enhance tau clearance via specific intracellular degradation pathways.

Genomic Epidemiology and Antimicrobial Susceptibility Profile of Enterotoxigenic Escherichia coli From Outpatients With Diarrhea in Shenzhen, China, 2015-2020.

Enterotoxigenic Escherichia coli (ETEC) is the leading cause of severe diarrhea in children and the most common cause of diarrhea in travelers. However, most ETEC infections in Shenzhen, China were from indigenous adults. In this study, we characterized 106 ETEC isolates from indigenous outpatients with diarrhea (77% were adults aged >20 years) in Shenzhen between 2015 and 2020 by whole-genome sequencing and antimicrobial susceptibility testing. Shenzhen ETEC isolates showed a remarkable high diversity, which belonged to four E. coli phylogroups (A: 71%, B1: 13%, E: 10%, and D: 6%) and 15 ETEC lineages, with L11 (25%, O159:H34/O159:H43, ST218/ST3153), novel L2/4 (21%, O6:H16, ST48), and L4 (15%, O25:H16, ST1491) being major lineages. Heat-stable toxin (ST) was most prevalent (76%, STh: 60% STp: 16%), followed by heat-labile toxin (LT, 17%) and ST + LT (7%). One or multiple colonization factors (CFs) were identified in 68 (64%) isolates, with the common CFs being CS21 (48%) and CS6 (34%). Antimicrobial resistance mutation/gene profiles of genomes were concordant with the phenotype testing results of 52 representative isolates, which revealed high resistance rate to nalidixic acid (71%), ampicillin (69%), and ampicillin/sulbactam (46%), and demonstrated that the novel L2/4 was a multidrug-resistant lineage. This study provides novel insight into the genomic epidemiology and antimicrobial susceptibility profile of ETEC infections in indigenous adults for the first time, which further improves our understanding on ETEC epidemiology and has implications for the development of vaccine and future surveillance and prevention of ETEC infections.

Concise solid-phase synthesis enables derivatisation of YEATS domain cyclopeptide inhibitors for improved cellular uptake.

YEATS domains, which are newly identified epigenetic readers of histone lysine acetylation and crotonylation, have emerged as promising anti-cancer drug targets. We recently developed AF9 YEATS domain-selective cyclopeptide inhibitors. However, the cumbersome and time-consuming synthesis of the cyclopeptides limited further structural derivatisation and applications. Here, we reported a concise method for the solid-phase synthesis of the cyclopeptides, which substantially reduced the amount of time required for the preparation of the cyclopeptides and led to a higher overall yield. Moreover, this new synthetic route also allowed further derivatisation of the cyclopeptides with various functional modules, including fluorescent dye and cell-penetrating peptide. We demonstrated that the conjugation of the cyclopeptide with cell-penetrating peptide TAT led to a significantly increased cellular uptake.

Comparative evaluation of six nucleic acid amplification kits for SARS-CoV-2 RNA detection.

BACKGROUND: SARS-CoV-2 is a newly emerged coronavirus, causing the coronavirus disease 2019 (COVID-19) outbreak in December, 2019. As drugs and vaccines of COVID-19 remain in development, accurate virus detection plays a crucial role in the current public health crisis. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) kits have been reliably used for detection of SARS-CoV-2 RNA since the beginning of the COVID-19 outbreak, whereas isothermal nucleic acid amplification-based point-of-care automated kits have also been considered as a simpler and rapid alternative. However, as these kits have only been developed and applied clinically within a short timeframe, their clinical performance has not been adequately evaluated to date. We describe a comparative study between a newly developed cross-priming isothermal amplification (CPA) kit (Kit A) and five RT-qPCR kits (Kits B-F) to evaluate their sensitivity, specificity, predictive values and accuracy. METHODS: Fifty-two clinical samples were used including throat swabs (n = 30), nasal swabs (n = 7), nasopharyngeal swabs (n = 7) and sputum specimens (n = 8), comprising confirmed (n = 26) and negative cases (n = 26). SARS-CoV-2 detection was simultaneously performed on each sample using six nucleic acid amplification kits. The sensitivity, specificity, positive/negative predictive values (PPV/NPV) and the accuracy for each kit were assessed using clinical manifestation and molecular diagnoses as the reference standard. Reproducibility for RT-qPCR kits was evaluated in triplicate by three different operators using a SARS-CoV-2 RNA-positive sample. On the basis of the six kits' evaluation results, CPA kit (Kit A) and two RT-qPCR Kits (Kit B and F) were applied to the SARS-CoV-2 detection in close-contacts of COVID-19 patients. RESULTS: For Kit A, the sensitivity, specificity, PPV/NPV and accuracy were 100%. Among the five RT-qPCR kits, Kits B, C and F had good agreement with the clinical diagnostic reports (Kappa ≥ 0.75); Kits D and E were less congruent (0.4 ≤ Kappa < 0.75). Differences between all kits were statistically significant (P < 0.001). The reproducibility of RT-qPCR kits was determined using a coefficients of variation (CV) between 0.95% and 2.57%, indicating good reproducibility. CONCLUSIONS: This is the first comparative study to evaluate CPA and RT-qPCR kits' specificity and sensitivity for SARS-CoV-2 detection, and could serve as a reference for clinical laboratories, thus informing testing protocols amid the rapidly progressing COVID-19 pandemic.

A Novel Molecular Method for Simultaneous Identification of Vibrio parahaemolyticus 57 K-Serogroups Using Probe Melting Curve Analysis.

The serotyping of Vibrio parahaemolyticus, which is crucial to the surveillance and detection of outbreaks of vibriosis infection, has been widely used in many countries. In this study, we developed a molecular assay, named multiplex ligation reaction based on probe melting curve analysis (MLMA), for simultaneous identification of V. parahaemolyticus 57 K-serogroups. Based on the previous genomes of 418 strains including 39 K-serogroups and the 18 K-serogroups sequences from public databases, we obtained 57 K-serogroups specific gene sequences for designing primers and probes. The developed MLMA assay for identifying the V. parahaemolyticus 57 K-serogroups showed high reproducibility, with the intra- and inter-assay standard deviations and coefficients of variation of no more than 1°C and 1%, respectively. The limit of detection for all gene targets ranged from 0.1 to 1.0 ng/µl. We validated the MLMA assay with a double-blind test identifying 595 V. parahaemolyticus isolates using conventional serotyping methods for comparison. The results showed the kappa value between the MLMA assay and the traditional serological method was 0.936 and that there was a 96.97% consistency rate with conventional serotyping methods for all detected isolates. Additionally, five rare K-serogroups were identified using the MLMA assay, as well as 18 strains that could not be identified using the traditional serotyping method. Thus, the MLMA assay provides a rapid, robust, and promising tool for the molecular serotyping of V. parahaemolyticus K-serogroups and has the potential application to the detection of outbreaks and surveillance of V. parahaemolyticus infection.

Molecular design of stapled pentapeptides as building blocks of self-assembled coiled coil-like fibers.

Peptide self-assembly inspired by natural superhelical coiled coils has been actively pursued but remains challenging due to limited helicity of short peptides. Side chain stapling can strengthen short helices but is unexplored in design of self-assembled helical nanofibers as it is unknown how staples could be adapted to coiled coil architecture. Here, we demonstrate the feasibility of this design for pentapeptides using a computational method capable of predicting helicity and fiber-forming tendency of stapled peptides containing noncoded amino acids. Experiments showed that the best candidates, which carried an aromatically substituted staple and phenylalanine analogs, displayed exceptional helicity and assembled into nanofibers via specific head-to-tail hydrogen bonding and packing between staple and noncoded side chains. The fibers exhibited sheet-of-helix structures resembling the recently found collapsed coiled coils whose formation was sensitive to side chain flexibility. This study expands the chemical space of coiled coil assemblies and provides guidance for their design.

Apparent diffusion coefficient values of cryptorchid testes and malignant transformation of cryptorchidism (MTC) (seminoma) in postpubertal patients.

OBJECTIVES: Diffusion-weighted imaging signal contrast can be quantified by apparent diffusion coefficient (ADC) maps, which reflect the diffusion properties of the examined tissue and are helpful for identifying pathology. To determine ADC values of cryptorchid testes in post-pubertal patients and assess performance for characterizing cryptorchid testes. METHODS: The medical records from 35 patients with unilateral scrotal vacuity were retrospectively reviewed. Data were analyzed in three groups: Group A, normal testes (i.e. the contralateral testes of the patients with cryptorchidism or MTC); Group B, cryptorchid testes; and Group C, malignant transformation of cryptorchidism (MTC) (seminoma). DWI used b-values of 0 and 800 s/mm2. Mean ADC values were compared using the independent samples t-test. The ability of ADC values was assessed using receiver operating characteristic curve analysis. The sensitivity, specificity, and accuracy were calculated. RESULTS: Mean ADC values for normal testes, cryptorchid testes, and MTC were 1.18 ± 0.18×10-3 mm2/s, 1.82 ± 0.40×10-3 mm2/s, and 0.80 ± 0.06×10-3 mm2/s, respectively. There were statistically significant differences in mean ADC values between normal testes and cryptorchid testes or MTC (p < 0.001). The cut-off ADC value for differentiating normal testes from cryptorchid testes was 1.47 × 10-3 mm2/s. The sensitivity, specificity, and accuracy were 88%, 91%, and 90%, respectively. The cut-off ADC value for differentiating normal testes from MTC was 1.22 × 10-3 mm2/s. The sensitivity, specificity, and accuracy were 100%, 31%, and 43%, respectively. CONCLUSION: ADC values of cryptorchid testes may be used to inform clinical decision-making and also monitor testicular function in patients who retain undescended testicles or post-operatively. ADVANCES IN KNOWLEDGE: Mean ADC values of cryptorchidism and MTC (seminoma) were used to reflect their pathological features.

Selective Targeting of AF9 YEATS Domain by Cyclopeptide Inhibitors with Preorganized Conformation.

YEATS domains are newly identified epigenetic "readers" of histone lysine acetylation (Kac) and crotonylation (Kcr). The malfunction of YEATS-Kac/Kcr interactions has been found to be involved in the pathogenesis of human diseases, such as cancer. These discoveries suggest that the YEATS domains are promising novel drug targets. We and others recently reported the development of YEATS domain inhibitors. Although these inhibitors have a general preference toward the AF9 and ENL YEATS domains, selective inhibitors targeting either YEATS domain are challenging to develop as these two proteins share a high structural similarity. In this study, we identified a proximal site outside the acyllysine-binding pocket that can differentiate AF9 YEATS from ENL YEATS. Combinatorial targeting of both the acyllysine pocket and this additional site by conformationally preorganized cyclopeptides enabled the selective inhibition of the AF9 YEATS domain. The most selective inhibitor, JYX-3, showed a 38-fold higher binding affinity toward AF9 YEATS over ENL YEATS. Further investigations indicated that JYX-3 could engage with AF9 in living cells, disrupt the YEATS-dependent chromatin recruitment of AF9, and suppress the transcription of AF9 target genes.