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Abstract Cryptococcal meningitis is a high-mortality infection. Adding 5-fluorocytosine (5-FC) to its treatment improves outcomes, but resistance to 5-FC presents a significant challenge. We conducted whole-genome sequencing on seven C. neoformans isolates with varying 5-FC susceptibility, along with proteomic and in silico analyses. Our findings indicate that mutations in genes of the pyrimidine salvage pathway are responsible for 5-FC resistance. Specifically, we identified an E64G missense mutation in the FUR1 gene, a large deletion in the FCY1 gene, and a point mutation in FCY1 leading to a truncated protein. The proteomic data indicated that these mutations resulted in the absence or reduction of crucial enzymes in resistant isolates. Genetic transformations confirmed the association between these mutations and 5-FC resistance. Resistance to 5-FC can develop during treatment and is closely tied to mutations in key metabolic enzymes. Understanding in vivo resistance development is crucial for combating resistance and enhancing patient outcomes.

Zhenzhen Sun,1,&ast; Kun Wang,1,&ast; Chuan Yun,1 Fang Bai,1 Xiaodan Yuan,2 Yaujiunn Lee,3 Qingqing Lou1 1The First Affiliated Hospital of Hainan Medical University, Hainan Clinical Research Center for Metabolic Disease, Haikou, Hainan, People’s Republic of China; 2Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, People’s Republic of China; 3Department of Endocrinology, Lee’s Clinic, Pingtung City, Pingtung County, Taiwan&ast;These authors contributed equally to this workCorrespondence: Qingqing Lou, The First Affiliated Hospital of Hainan Medical University, Hainan Clinical Research Center for Metabolic Disease, No. 31, Longhua Road, Haikou, 570102, Hainan, People’s Republic of China, Tel +86 15312019129, Email 2444890144@qq.comPurpose: To investigate the association of five obesity indices and the variability of these indices with diabetic kidney disease (DKD) in patients with type 2 diabetes and compare the predictive validity of these markers for the risk of DKD in this large longitudinal cohort study.Patients and Methods: A total of 2659 patients with type 2 diabetes who did not have DKD were enrolled between 2006 and 2019 at Lee’s United Clinic in Taiwan. Data were collected for each subject, including demographic data, personal medical history, clinical parameters and calculated Body mass index (BMI), visceral adiposity index (VAI), lipid accumulation product (LAP), body roundness index (BRI) and variability of five obesity indices. Cox regression analysis was performed to determine the relationship between different obesity indicators and DKD risk. Cox’s proportional hazards model was evaluated the predictive effect of obesity indices on DKD.Results: The risk of developing DKD increased with an increase in the BRI, LAP, VAI, WC and BMI (all P trend< 0.05), and the variability of VAI was significantly associated with DKD [HR=1.132, 95% CI (1.001, 1.281)] after adjusting for corresponding variables. BRI had the strongest predictive effect on DKD. BRI had the best predictive performance, with AUC of 0.807, 0.663 and 0.673 at 1, 3 and 5 years, respectively. Cox regression analysis of risk factors for DKD in patients stratified by BRI quartiles showed that patients in the Q4 group had the highest risk of developing DKD [HR=1.356, 95% CI (1.131, 1.626)].Conclusion: BMI, WC, VAI, LAP, BRI and VAI variability were associated with a significant increase in the risk of DKD events, and BRI was superior and alternative obesity index for predicting DKD.Keywords: visceral adiposity index, lipid accumulation product, body roundness index, diabetic kidney disease

Abstract Circulating miR-371a-3p has excellent performance in the detection of viable (non-teratoma) germ cell tumor (GCT) pre-orchiectomy; however, its ability to detect occult disease is understudied. To refine the serum miR-371a-3p assay in the minimal residual disease setting we compared performance of raw (Cq) and normalized (∆Cq, RQ) values from prior assays, and validated interlaboratory concordance by aliquot swapping. Revised assay performance was determined in a cohort of 32 patients suspected of occult retroperitoneal disease. Assay superiority was determined by comparing resulting receiver-operator characteristic (ROC) curves using the Delong method. Pairwise t-tests were used to test for interlaboratory concordance. Performance was comparable when thresholding based on raw Cq vs. normalized values. Interlaboratory concordance of miR-371a-3p was high, but reference genes miR-30b-5p and cel-miR-39-3p were discordant. Introduction of an indeterminate range of Cq 28–35 with a repeat run for any indeterminate improved assay accuracy from 0.84 to 0.92 in a group of patients suspected of occult GCT. We recommend that serum miR-371a-3p test protocols are updated to (a) utilize threshold-based approaches using raw Cq values, (b) continue to include an endogenous (e.g., miR-30b-5p) and exogenous non-human spike-in (e.g., cel-miR-39-3p) microRNA for quality control, and (c) to re-run any sample with an indeterminate result.

Flow cytometry is commonly employed for ploidy determination and cell cycle analysis in cryptococci. The cells are subjected to fixation and staining with DNA-binding fluorescent dyes, most commonly with propidium iodide (PI), before undergoing flow cytometric analysis. In ploidy determination, cell populations are classified according to variations in DNA content, as evidenced by the fluorescence intensity of stained cells. As reported in Saccharomyces cerevisiae, we found drawbacks with PI staining that confounded the accurate analysis of ploidy by flow cytometry when the size of the cryptococci changed significantly. However, the shift in the fluorescence intensity, unrelated to ploidy changes in cells with increased size, could be accurately interpreted by applying the ImageStream system. SYTOX Green or SYBR Green I, reported to enable DNA analysis with a higher accuracy than PI in S. cerevisiae, were nonspecific for nuclear DNA staining in cryptococci. Until dyes or methods capable of reducing the variability inherent in the drastic changes in cell size or shape become available, PI appears to remain the most reliable method for cell cycle or ploidy analysis in Cryptococcus.

ABSTRACT Flucytosine (5-FC) is an antifungal agent commonly used for treatment of cryptococcosis and several other systemic mycoses. In fungi, cytosine permease and cytosine deaminase are known major players in flucytosine resistance by regulating uptake and deamination of 5-FC, respectively. Cryptococcus species have three paralogs each of cytosine permease (FCY2, FCY3, and FCY4) and cytosine deaminase (FCY1, FCY5 and FCY6). As in other fungi, we found FCY1 and FCY2 to be the primary cytosine deaminase and permease gene, respectively, in C. neoformans H99 (VNI), C. gattii R265 (VGIIa) and WM276 (VGI). However, when various amino acids were used as the sole nitrogen source, C. neoformans and C. gattii diverged in the function of FCY3 and FCY6. Though there was some lineage-dependent variability, the two genes functioned as the secondary permease and deaminase, respectively, only in C. gattii when the nitrogen source was arginine, asparagine, or proline. Additionally, the expression of FCY genes, excluding FCY1, was under nitrogen catabolic repression in the presence of NH4. Functional analysis of GAT1 and CIR1 gene deletion constructs demonstrated that these two genes regulate the expression of each permease and deaminase genes individually. Furthermore, the expression levels of FCY3 and FCY6 under different amino acids corroborated the 5-FC susceptibility in fcy2Δ or fcy1Δ background. Thus, the mechanism of 5-FC resistance in C. gattii under diverse nitrogen conditions is orchestrated by two transcription factors of GATA family, cytosine permease and deaminase genes. IMPORTANCE 5-FC is a commonly used antifungal drug for treatment of cryptococcosis caused by Cryptococcus neoformans and C. gattii species complexes. When various amino acids were used as the sole nitrogen source for growth, we found lineage dependent differences in 5-FC susceptibility. Deletion of the classical cytosine permease (FCY2) and deaminase (FCY1) genes caused increased 5-FC resistance in all tested nitrogen sources in C. neoformans but not in C. gattii. Furthermore, we demonstrate that the two GATA family transcription factor genes GAT1 and CIR1 are involved in the nitrogen-source dependent 5-FC resistance by regulating the expression of the paralogs of cytosine permease and deaminase genes. Our study not only identifies the new function of paralogs of the cytosine permease and deaminase and the role of their regulatory transcription factors but also denotes the differences in the mechanism of 5-FC resistance among the two etiologic agents of cryptococcosis under different nitrogen sources.

Enzyme-Linked Immunosorbent Assay (ELISA) is a versatile and widely used technique to detect and quantify small molecules in various clinical and analytical applications. It is also an effective tool for demonstrating the specificity between antibody and antigen to students across diverse disciplines. However, undergraduate laboratories often face the difficulty of performing proper ELISA exercise due to time and facility constraints. Here, we report a cost-effective, technically simple ELISA exercise which can be completed within 2.5-h at room temperature. This microtitre plate-based ELISA exercise utilises easily sourced inexpensive materials and simulated serum samples which are safe to handle in the undergraduate lab settings. The hands-on procedure and a real-life scenario of disease (Rheumatoid Arthritis) diagnosis reinforce students' understanding of antigen-antibody interactions. ELISA results are displayed by a colourimetric enzymatic reaction, which can be analysed by eye without specialised equipment. This laboratory exercise was designed as part of the final year's immunology course in a three-year undergraduate Biomedical Science programme, but can also be adapted to suit a broad range of student skill levels in science-fields because of its easy set-up and low technical requirement. The procedure and scenario can be also adjusted to meet the specific needs of other educator's goals.

Detailed characterization of the cell types in the human brain requires scalable experimental approaches to examine multiple aspects of the molecular state of individual cells, as well as computational integration of the data to produce unified cell-state annotations. Here we report improved high-throughput methods for single-nucleus droplet-based sequencing (snDrop-seq) and single-cell transposome hypersensitive site sequencing (scTHS-seq). We used each method to acquire nuclear transcriptomic and DNA accessibility maps for >60,000 single cells from human adult visual cortex, frontal cortex, and cerebellum. Integration of these data revealed regulatory elements and transcription factors that underlie cell-type distinctions, providing a basis for the study of complex processes in the brain, such as genetic programs that coordinate adult remyelination. We also mapped disease-associated risk variants to specific cellular populations, which provided insights into normal and pathogenic cellular processes in the human brain. This integrative multi-omics approach permits more detailed single-cell interrogation of complex organs and tissues.

Lihua Wang,1,&ast; Canhua Yun,2,&ast; Fengyan Huang,1 Juan Xiao,3 Yanli Ju,1 Fang Cheng,1 Wei Zhang,2 Hongying Jia1,3 1Department of Biostatistics, School of Public Health, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, 250012, People’s Republic of China; 2Department of Nuclear Medicine, the Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, 250033, People’s Republic of China; 3Center of Evidence-Based Medicine, Institute of Medical Sciences, the Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, 250033, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Hongying JiaDepartment of Biostatistics, School of Public Health, Cheeloo College of Medicine, Shandong University, No. 44 Wenhua West Road, Jinan, Shandong, 250012, People’s Republic of ChinaTel +86 17660089053Email jiahongying@sdu.edu.cnWei ZhangDepartment of Nuclear Medicine, the Second Hospital, Cheeloo College of Medicine, Shandong University, No. 247 Beiyuan Street, Jinan, Shandong, 250033, People’s Republic of ChinaTel +86 17660086070Email sdeyzhangwei@163.comPurpose: The aim of this study was to evaluate the value of preablative stimulated thyroglobulin (presTg) and thyroglobulin reduction index (TRI) to predict the different responses to second radioactive iodine (RAI) therapy in differentiated thyroid cancer (DTC) patients with structural incomplete response (SIR).Patients and Methods: A single-center retrospective study analyzed the different clinical outcomes after second RAI therapy in 206 patients with SIR. PresTg1 and presTg2 were measured before first and second RAI management and TRI was the reduction index of presTg1 and presTg2. Cut-off values of presTg and TRI were obtained using receiver operating characteristic analysis. The univariate logistic regression analysis was performed to confirm these parameters as prognostic factors to predict different responses to second RAI therapy.Results: Only ATA risk stratification, the post-therapy whole-body scanning (Rx-WBS) findings, presTg1, presTg2, TRI, were different in patients with SIR. After second RAI therapy, 28.2% (58/206) of patients with SIR initially were reclassified as excellent response (ER). PresTg1 < 6.6 ng/mL, presTg2 < 1.2ng/mL, and TRI > 74.2% were excellent indications to predict ER from non-ER after second RAI treatment. PresTg1 > 14.9 ng/mL, presTg2 > 1.8ng/mL and TRI < 66.5% were well markers to predict poor outcome (SIR). High risk and distant metastases could still be considered as risk factors.Conclusion: DTC patients with SIR could benefit through second RAI treatment. PresTg before each RAI therapy and TRI could be considered as effective decision-making markers for second RAI therapy and as predictive indications for clinical outcomes.Keywords: structural incomplete response, radioactive iodine therapy, preablative stimulated thyroglobulin, thyroglobulin reduction index, differentiated thyroid carcinoma

Jingjia Cao, Canhua Yun, Xiaolu Zhu, Xiao Li, Yaru Sun, Wei Zhang Department of Nuclear Medicine, The Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan, 250033, People’s Republic of ChinaCorrespondence: Wei ZhangDepartment of Nuclear Medicine, The Second Hospital, Cheeloo College of Medicine, Shandong University, No. 247 Road, Tianqiao District, Jinan, 250033, People’s Republic of ChinaTel +86 17660086070Email sdeyzhangwei@163.comPurpose: To explore the factors that influence the short-term clinical outcome after the first 131I treatment of papillary thyroid micro carcinoma (PTMC).Patients and Methods: From October 2015 to June 2018, patients who were diagnosed with PTMC with lymph node metastasis were analyzed retrospectively, excluding patients with incomplete clinical data, distant metastasis, positive TGAb, TSH< 30 mIU/L. The baseline data of sex, age, time from last surgery to first 131I treatment, tumor pathology information, and biochemical information were collected before admission. All patients included had radioactive iodine (RAI) with 3.70 GBq. The treatment response of patients was evaluated 6– 8 months after discharge. By means of univariate and multivariate analysis, including excellent response (ER) and non-excellent response (NER) groups of clinical data, we assessed the impact of 131I on patients’ outcome. A nomogram model was established based on the above independent risk factors.Results: A total of 206 patients (59 males and 147 females, mean age 43.4 ± 10.6 years) were included in the study. The median follow-up time was 169.4 ± 10.5 days, including 139 patients in ER group (67.4%) and 67 patients in NER group (32.5%). Four factors including combining Hashimoto’s thyroiditis, pre-ablative Tg levels, UIE levels, and lateral lymph node numbers were statistically different between ER group and NER group with significance at P < 0.05. Further multivariate analysis showed that Hashimoto’s thyroiditis and Ps-Tg levels could be used as independent factors. The model verification showed that the C-index of the modeling set was 0.822, indicating that the nomogram model had a good predicted accuracy.Conclusion: Our data suggest that coexisting Hashimoto’s thyroiditis and elevated Ps-Tg levels are predictive factors for short-term outcome of thyroid micro papillary carcinoma after 131I treatment. Also, the nomogram model had a good predicted accuracy.Keywords: micro papillary carcinoma, curative effect, 131I treatment, outcome

Pathogenic fungi rapidly develop resistance to the antifungal agent 5-fluorocytosine (5-FC). Here, Chang et al. explore the mechanisms by which Cryptococcus develops 5-FC resistance at a high frequency, including mutations in several genes and altered levels of key metabolites.

Peng Su,1,2 Jun-lin Zhou,2 Cai Yun,1 Feng Liu,1 Yi Zhang1 1Department of Orthopaedics, Teaching Hospital of Capital Medical University, Beijing Shijingshan Hospital, Beijing, People’s Republic of China; 2Department of Orthopedics, Beijing Chaoyang Hospital, Capital Medical University, Beijing, People’s Republic of ChinaCorrespondence: Jun-lin ZhouDepartment of Orthopedics, Beijing Chaoyang Hospital, Capital Medical University, 8 Gongren Tiyuchang Nanlu, Chaoyang District, Beijing, 100020, People’s Republic of ChinaTel +86-13240718193Email zhoujunlin145@163.comObjective: This study aimed to directly and accurately measure the range of motion of the acromioclavicular joint through 3D reconstruction and image registration.Methods: Thirteen healthy volunteers participated in the study. Computerized tomography (CT) was used to measure the acromioclavicular joint in four different motion poses. The images were integrated using reconstruction and registration technology, and the rotation angle range of the acromioclavicular joint was measured using 3D reconstruction. The measurements were expressed by axial angle representation. The dominant and the non-dominant sides were compared, and the difference in the axial angle of the acromioclavicular joint was compared in different postures.Results: The difference between the dominant and non-dominant sides in acromioclavicular motion was not significant. In the sagittal motion of the upper limb, the rotation angle of the acromioclavicular joint was greatest in a resting horizontal position, with an average of 26.1°. In this position, 34.6% of flexion and extension movements of the upper limb were completed by the acromioclavicular joint, which was the highest level of engagement. The rotation angle was lowest in the resting posteroinferior position, with an average of 8.2°. In this position, 24.9% of the flexion and extension movements of the upper limb were completed by the acromioclavicular joint.Conclusion: Combining 3D reconstruction and image registration is a direct and accurate method of measuring the motion of the acromioclavicular joint. The rotational motions of the acromioclavicular joint are bilaterally symmetrical and can be used as a reference for comparative study. The maximum range of motion of the acromioclavicular joint during rotation was found in the resting horizontal position. The clinical examination and post-treatment evaluation of the acromioclavicular joint’s rotation function can therefore be targeted in this range.Keywords: image registration, axial angle representation, acromioclavicular joints

Significant heterogeneities in gene expression among individual cells are typically interrogated using single whole cell approaches. However, tissues that have highly interconnected processes, such as in the brain, present unique challenges. Single-nucleus RNA sequencing (SNS) has emerged as an alternative method of assessing a cell's transcriptome through the use of isolated nuclei. However, studies directly comparing expression data between nuclei and whole cells are lacking. Here, we have characterized nuclear and whole cell transcriptomes in mouse single neurons and provided a normalization strategy to reduce method-specific differences related to the length of genic regions. We confirmed a high concordance between nuclear and whole cell transcriptomes in the expression of cell type and metabolic modeling markers, but less so for a subset of genes associated with mitochondrial respiration. Therefore, our results indicate that single-nucleus transcriptome sequencing provides an effective means to profile cell type expression dynamics in previously inaccessible tissues.

Cryptococcosis is a potentially lethal disease of humans/animals caused by Cryptococcus neoformans and Cryptococcus gattii. Distinction between the two species is based on phenotypic and genotypic characteristics. Recently, it was proposed that C. neoformans be divided into two species and C. gattii into five species based on a phylogenetic analysis of 115 isolates. While this proposal adds to the knowledge about the genetic diversity and population structure of cryptococcosis agents, the published genotypes of 2,606 strains have already revealed more genetic diversity than is encompassed by seven species. Naming every clade as a separate species at this juncture will lead to continuing nomenclatural instability. In the absence of biological differences between clades and no consensus about how DNA sequence alone can delineate a species, we recommend using "Cryptococcus neoformans species complex" and "C. gattii species complex" as a practical intermediate step, rather than creating more species. This strategy recognizes genetic diversity without creating confusion.

Lungs balance threat from primary viral infection, secondary infection, and inflammatory damage. Severe pulmonary inflammation induces vascular permeability, edema, and organ dysfunction. We previously demonstrated that poly(I:C) (pICLC) induced type 1 interferon (t1IFN) protected mice from Cryptococcus gattii (Cg) via local iron restriction. Here we show pICLC increased serum protein and intravenously injected FITC-dextran in the lung airspace suggesting pICLC induces vascular permeability. Interestingly, pICLC induced a pro-inflammatory signature with significant expression of IL-1 and IL-6 which depended on MDA5 and t1IFN. Vascular permeability depended on MDA5, t1IFN, IL-1, and IL-6. T1IFN also induced MDA5 and other MDA5 signaling components suggesting that positive feedback contributes to t1IFN dependent expression of the pro-inflammatory signature. Vascular permeability, induced by pICLC or another compound, inhibited Cg by limiting iron. These data suggest that pICLC induces t1IFN which potentiates pICLC-MDA5 signaling increasing IL-1 and IL-6 resulting in leakage of antimicrobial serum factors into lung airspace. Thus, induced vascular permeability may act as an innate defense mechanism against opportunistic fungal infection, such as cryptococcosis, and may be exploited as a host-directed therapeutic target.

Canhua Yun,1 Meiling Wu,1 Juan Xiao,2 Yong Liu,1 Wei Zhang,1 Jingjia Cao1 1Department of Nuclear Medicine, The Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan, 250033, People’s Republic of China; 2Center of Evidence-Based Medicine, Institute of Medical Sciences, The Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan, 250033, People’s Republic of ChinaCorrespondence: Jingjia Cao No. 247, Beiyuan Street, Jinan City, Shandong Province, People’s Republic of ChinaTel +86 17660083825Email sdhyx1990@163.comObjective: To compare the efficacy and influencing factors of initial radioactive iodine (RAI) ablation therapy for postoperative N1 stage papillary thyroid micro-carcinoma (PTMC) and papillary thyroid non-micro carcinoma (PTC), and to explore the necessity of RAI for N1 stage PTMC.Methods: A retrospective analysis of patients with N1 stage papillary thyroid cancer who underwent RAI in our department from January 2018 to June 2019. According to the tumor diameter, papillary thyroid carcinoma was divided into PTMC group (≤ 1.0cm) with 129 patients and PTC group (> 1.0 cm) with 214 patients. According to the 2015 ATA guidelines, the patient’s treatment response was evaluated 6– 8 months after discharge from the hospital: excellent response (ER), indeterminate response (IDR), biochemical incomplete response (BIR), and structural incomplete response (SIR). IDR, BIR, and SIR were classified into NER group. Chi-squared test, independent sample t-test, Mann–Whitney U test, and binary logistic regression analysis were used to compare the differences between PTMC and PTC patients.Results: The ps-Tg of the PTMC group was significantly lower than that of the PTC group (P = 0.001), and the ER ratio of the PTMC group was higher (χ2 = 5.445, P < 0.05). The ER ratio of PTMC patients in the N1a group was significantly higher than that of PTC patients (80%, 66.7%, χ2 = 4.076, P < 0.05), while the ER ratio of PTMC in the N1b group was not significantly different from that of PTC. Gender, N stage, and ps-Tg were found to be independent factors of RAI treatment response.Conclusion: The efficacy of the initial RAI of PTMC patients was significantly better than that of PTC patients. There was no significant difference in the efficacy of RAI between males with PTMC, N1b stage, ps-Tg ≥ 5.87ng/mL and PTC patients, which suggested that RAI is necessary for these patients.Keywords: papillary thyroid micro-carcinoma, papillary thyroid non-micro carcinoma, curative effect, RAI

Lysophosphatidic acid (LPA) is a bioactive lipid molecule that regulates a wide array of cellular functions, including proliferation, differentiation, and survival, via activation of cognate receptors. The LPA5 receptor is highly expressed in the intestinal epithelium, but its function in restoring intestinal epithelial integrity following injury has not been examined. Here, we use a radiation-induced injury model to study the role of LPA5 in regulating intestinal epithelial regeneration. Control mice (Lpar5f/f) and mice with an inducible, epithelial cell-specific deletion of Lpar5 in the small intestine (Lpar5IECKO) were subjected to 10 Gy total body X-ray irradiation and analyzed during recovery. Repair of the intestinal mucosa was delayed in Lpar5IECKO mice with reduced epithelial proliferation and increased crypt cell apoptosis. These effects were accompanied by reduced numbers of OLFM4+ intestinal stem cells (ISCs). The effects of LPA5 on ISCs were corroborated by studies using organoids derived from Lgr5-lineage tracking reporter mice with deletion of Lpar5 in Lgr5+-stem cells (Lgr5Cont or Lgr5ΔLpar5). Irradiation of organoids resulted in fewer numbers of Lgr5ΔLpar5 organoids retaining Lgr5+-derived progenitor cells compared with Lgr5Cont organoids. Finally, we observed that impaired regeneration in Lpar5IECKO mice was associated with reduced numbers of Paneth cells and decreased expression of Yes-associated protein (YAP), a critical factor for intestinal epithelial repair. Our study highlights a novel role for LPA5 in regeneration of the intestinal epithelium following irradiation and its effect on the maintenance of Paneth cells that support the stem cell niche. NEW & NOTEWORTHY: We used mice lacking expression of the lysophosphatidic acid receptor 5 (LPA5) in intestinal epithelial cells and intestinal organoids to show that the LPA5 receptor protects intestinal stem cells and progenitors from radiation-induced injury. We show that LPA5 induces YAP signaling and regulates Paneth cells. [ABSTRACT FROM AUTHOR]

The human brain has enormously complex cellular diversity and connectivities fundamental to our neural functions, yet difficulties in interrogating individual neurons has impeded understanding of the underlying transcriptional landscape. We developed a scalable approach to sequence and quantify RNA molecules in isolated neuronal nuclei from a postmortem brain, generating 3227 sets of single-neuron data from six distinct regions of the cerebral cortex. Using an iterative clustering and classification approach, we identified 16 neuronal subtypes that were further annotated on the basis of known markers and cortical cytoarchitecture. These data demonstrate a robust and scalable method for identifying and categorizing single nuclear transcriptomes, revealing shared genes sufficient to distinguish previously unknown and orthologous neuronal subtypes as well as regional identity and transcriptomic heterogeneity within the human brain.

The transcriptional state of a cell reflects a variety of biological factors, from cell-type-specific features to transient processes such as the cell cycle, all of which may be of interest. However, identifying such aspects from noisy single-cell RNA-seq data remains challenging. We developed pathway and gene set overdispersion analysis (PAGODA) to resolve multiple, potentially overlapping aspects of transcriptional heterogeneity by testing gene sets for coordinated variability among measured cells.

This paper reported antibiotic resistance and PFGE genetic typing of Streptococcus thermophilus stains isolated from different yogurt brands in Chinese market. In total, 42 strains isolated from 43 yogurt samples were identified as S. thermophilus stains by 16S rDNA sequence analysis. The investigation of antibiotic-resistance profiles revealed that those isolates were resistant to erythromycin (69.05 %), clindamycin (73.81 %), chloramphenicol (61.90 %) and fosfomycin (19.05 %), and were susceptible to other 13 kinds of antibiotics. The genetic typing of S. thermophilus stains was examined by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA digested with SmaI. The enzyme restriction profiles showed the 42 S. thermophilus isolates were divided into 14 PFGE types, and further analysis showed the PFGE profile did not completely match with antibiotic resistance profile. The antibiotic resistance and PFGE pattern database generated in this study suggest that the safety evaluation of S. thermophilus should be paid more attention and will provide basic for information of food safe assessment of yogurt.

ABSTRACT The Cryptococcus gattii species complex has often been referred to as a primary pathogen due to its high infection frequency among apparently immunocompetent patients. In order to scrutinize the immune status of patients and the lineages of etiologic agents, we analyzed patient histories and the molecular types of etiologic agents from 135 global C. gattii cases. Eighty-six of 135 patients had been diagnosed as immunocompetent, although some of them had underlying medical issues, and 49 were diagnosed as immunocompromised with risk factors similar to those seen in Cryptococcus neoformans infection. We focused on the 86 apparently immunocompetent patients and were able to obtain plasma from 32 (37%) to analyze for the presence of autoantibodies against the granulocyte-macrophage colony-stimulating factor (GM-CSF) since these antibodies have been reported as a hidden risk factor for C. gattii infection. Among the 32 patients, 25 were free from any known other health issues, and 7 had various medical conditions at the time of diagnosis for cryptococcosis. Importantly, plasma from 19 (76%) of 25 patients with no recognized underlying medical condition showed the presence of GM-CSF autoantibodies, supporting this antibody as a major hidden risk factor for C. gattii infection. These data indicate that seemingly immunocompetent people with C. gattii infection warrant detailed evaluation for unrecognized immunologic risks. There was no relationship between molecular type and underlying conditions of patients. Frequency of each molecular type was related to its geographic origin exemplified by the overrepresentation of VGIV in HIV-positive (HIV+) patients due to its prevalence in Africa. IMPORTANCE The C. neoformans and C. gattii species complex causes cryptococcosis. The C. neoformans species complex is known as an opportunistic pathogen since it primarily infects immunocompromised patients. C. gattii species complex has been referred to as a primary pathogen due to its high infection frequency in apparently immunocompetent people. We analyzed 135 global cases of C. gattii infection with documented patient history. Eighty-six of 135 patients were originally diagnosed as immunocompetent and 49 as immunosuppressed with similar underlying conditions reported for C. neoformans infection. A significant number of C. gattii patients without known underlying conditions possessed autoantibodies against granulocytes-macrophage colony-stimulating factor (GM-CSF) in their plasma, supporting the presence of GM-CSF antibodies as a hidden risk factor for C. gattii infection. No relationship was found between C. gattii lineages and the underlying conditions except for overrepresentation of the molecular type VGIV among HIV+ patients due to the prevalence of VGIV in Africa.