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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.

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.

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.

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

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

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.

ABSTRACT We discovered a new lineage of the globally important fungal pathogen Cryptococcus gattii on the basis of analysis of six isolates collected from three locations spanning the Central Miombo Woodlands of Zambia, Africa. All isolates were from environments (middens and tree holes) that are associated with a small mammal, the African hyrax. Phylogenetic and population genetic analyses confirmed that these isolates form a distinct, deeply divergent lineage, which we name VGV. VGV comprises two subclades (A and B) that are capable of causing mild lung infection with negligible neurotropism in mice. Comparing the VGV genome to previously identified lineages of C. gattii revealed a unique suite of genes together with gene loss and inversion events. However, standard URA5 restriction fragment length polymorphism (RFLP) analysis could not distinguish between VGV and VGIV isolates. We therefore developed a new URA5 RFLP method that can reliably identify the newly described lineage. Our work highlights how sampling understudied ecological regions alongside genomic and functional characterization can broaden our understanding of the evolution and ecology of major global pathogens. IMPORTANCE Cryptococcus gattii is an environmental pathogen that causes severe systemic infection in immunocompetent individuals more often than in immunocompromised humans. Over the past 2 decades, researchers have shown that C. gattii falls within four genetically distinct major lineages. By combining field work from an understudied ecological region (the Central Miombo Woodlands of Zambia, Africa), genome sequencing and assemblies, phylogenetic and population genetic analyses, and phenotypic characterization (morphology, histopathological, drug-sensitivity, survival experiments), we discovered a hitherto unknown lineage, which we name VGV (variety gattii five). The discovery of a new lineage from an understudied ecological region has far-reaching implications for the study and understanding of fungal pathogens and diseases they cause.

Abstract The Hydrocephalus Association Posthemorrhagic Hydrocephalus Workshop was held on July 25 and 26, 2016 at the National Institutes of Health. The workshop brought together a diverse group of researchers including pediatric neurosurgeons, neurologists, and neuropsychologists with scientists in the fields of brain injury and development, cerebrospinal and interstitial fluid dynamics, and the blood–brain and blood–CSF barriers. The goals of the workshop were to identify areas of opportunity in posthemorrhagic hydrocephalus research and encourage scientific collaboration across a diverse set of fields. This report details the major themes discussed during the workshop and research opportunities identified for posthemorrhagic hydrocephalus. The primary areas include (1) preventing intraventricular hemorrhage, (2) stopping primary and secondary brain damage, (3) preventing hydrocephalus, (4) repairing brain damage, and (5) improving neurodevelopment outcomes in posthemorrhagic hydrocephalus.

We demonstrate a broadband and long-distance intermediate frequency-over-fiber (IFoF) transmission scheme employing a transmitter composed of parallel intensity/phase (IM/PM) modulators with appropriate bandwidth allocations to IM and PM. Due to the proposed scheme, we can eliminate all the null frequencies caused by dispersion-induced RF power fading, which, in turn, enables us to significantly increase the available bandwidth. In addition, our system does not require any synchronization between IM and PM, which reduces complexity compared to conventional parallel transmitter architecture. We successfully transmitted 20 × 360 MHz filtered orthogonal-frequency-division-multiplexed signals corresponding to a common public radio interface equivalent data rate of 524.28 Gbps over a 30- and 40-km single-mode fiber satisfying the 8% threshold for the error-vector magnitude values for all the subcarriers. These results show that our proposed IFoF transmission scheme is scalable to long-distance mobile fronthaul links for 5G and beyond.

Abstract 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.

We design a heterogeneous 12-core 4-LP-mode fiber having two different types of cores and trench-assisted graded-index profiles. For the optimum design of the index profiles of these two heterogeneous cores, we first investigate the impacts of various geometric parameters on the effective refractive index of each LP mode, maximum differential modal delay (DMD), and bending losses of the guiding and higher order modes. We then design a heterogeneous 12-core 4-LP mode fiber having a cladding diameter of 242 μm. The numerical analyses based on the coupled-mode equation show that the inter-core crosstalk levels of the proposed fiber are lower than -55 dB/km, regardless of modes, as long as the fiber is not bent tightly with a bending radius of -3 μm-2 and 42.2, respectively.

ABSTRACT We found a novel role of Myo5, a type I myosin (myosin-I), and its fortuitous association with d-amino acid utilization in Cryptococcus gattii. Myo5 colocalized with actin cortical patches and was required for endocytosis. Interestingly, the myo5Δ mutant accumulated high levels of d-proline and d-alanine which caused toxicity in C. gattii cells. The myo5Δ mutant also accumulated a large set of substrates, such as membrane-permeant as well as non-membrane-permeant dyes, l-proline, l-alanine, and flucytosine intracellularly. Furthermore, the efflux rate of fluorescein was significantly increased in the myo5Δ mutant. Importantly, the endocytic defect of the myo5Δ mutant did not affect the localization of the proline permease and flucytosine transporter. These data indicate that the substrate accumulation phenotype is not solely due to a defect in endocytosis, but the membrane properties may have been altered in the myo5Δ mutant. Consistent with this, the sterol staining pattern of the myo5Δ mutant was different from that of the wild type, and the mutant was hypersensitive to amphotericin B. It appears that the changes in sterol distribution may have caused altered membrane permeability in the myo5Δ mutant, allowing increased accumulation of substrate. Moreover, myosin-I mutants generated in several other yeast species displayed a similar substrate accumulation phenotype. Thus, fungal type I myosin appears to play an important role in regulating membrane permeability. Although the substrate accumulation phenotype was detected in strains with mutations in the genes involved in actin nucleation, the phenotype was not shared in all endocytic mutants, indicating a complicated relationship between substrate accumulation and endocytosis. IMPORTANCE Cryptococcus gattii, one of the etiological agents of cryptococcosis, can be distinguished from its sister species Cryptococcus neoformans by growth on d-amino acids. C. gattii MYO5 affected the growth of C. gattii on d-amino acids. The myo5Δ cells accumulated high levels of various substrates from outside the cells, and excessively accumulated d-amino acids appeared to have caused toxicity in the myo5Δ cells. We provide evidence on the alteration of membrane properties in the myo5Δ mutants. Additionally, alteration in the myo5Δ membrane permeability causing higher substrate accumulation is associated with the changes in the sterol distribution. Furthermore, myosin-I in three other yeasts also manifested a similar role in substrate accumulation. Thus, while fungal myosin-I may function as a classical myosin-I, it has hitherto unknown additional roles in regulating membrane permeability. Since deletion of fungal myosin-I causes significantly elevated susceptibility to multiple antifungal drugs, it could serve as an effective target for augmentation of fungal therapy.

ABSTRACT Cryptococcus neoformans causes deadly mycosis primarily in AIDS patients, whereas Cryptococcus gattii infects mostly non-HIV patients, even in regions with high burdens of HIV/AIDS and an established environmental presence of C. gattii. As HIV induces type I IFN (t1IFN), we hypothesized that t1IFN would differentially affect the outcome of C. neoformans and C. gattii infections. Exogenous t1IFN induction using stabilized poly(I·C) (pICLC) improved murine outcomes in either cryptococcal infection. In C. neoformans-infected mice, pICLC activity was associated with C. neoformans containment and classical Th1 immunity. In contrast, pICLC activity against C. gattii did not require any immune factors previously associated with C. neoformans immunity: T, B, and NK cells, IFN-γ, and macrophages were all dispensable. Interestingly, C. gattii pICLC activity depended on β-2-microglobulin, which impacts iron levels among other functions. Iron supplementation reversed pICLC activity, suggesting C. gattii pICLC activity requires iron limitation. Also, pICLC induced a set of iron control proteins, some of which were directly inhibitory to cryptococcus in vitro, suggesting t1IFN regulates iron availability in the pulmonary air space fluids. Thus, exogenous induction of t1IFN significantly improves the outcome of murine infection by C. gattii and C. neoformans but by distinct mechanisms; the C. gattii effect was mediated by iron limitation, while the effect on C. neoformans infection was through induction of classical T-cell-dependent immunity. Together this difference in types of T-cell-dependent t1IFN immunity for different Cryptococcus species suggests a possible mechanism by which HIV infection may select against C. gattii but not C. neoformans. IMPORTANCE Cryptococcus neoformans and Cryptococcus gattii cause fatal infection in immunodeficient and immunocompetent individuals. While these fungi are sibling species, C. gattii infects very few AIDS patients, while C. neoformans infection is an AIDS-defining illness, suggesting that the host response to HIV selects C. neoformans over C. gattii. We used a viral mimic molecule (pICLC) to stimulate the immune response, and pICLC treatment improved mouse outcomes from both species. pICLC-induced action against C. neoformans was due to activation of well-defined immune pathways known to deter C. neoformans, whereas these immune pathways were dispensable for pICLC treatment of C. gattii. Since these immune pathways are eventually destroyed by HIV/AIDS, our data help explain why the antiviral immune response in AIDS patients is unable to control C. neoformans infection but is protective against C. gattii. Furthermore, pICLC induced tighter control of iron in the lungs of mice, which inhibited C. gattii, thus suggesting an entirely new mode of nutritional immunity activated by viral signals.

ABSTRACT Heteroresistance to fluconazole (FLC) in Cryptococcus is a transient adaptive resistance which is lost upon release from the drug pressure. It is known that clones heteroresistant to FLC invariably contain disomic chromosomes, but how disomy is formed remains unclear. Previous reports suggested that the aneuploid heteroresistant colonies in Cryptococcus emerge from multinucleated cells, resembling the case in Candida albicans. Although a small number of cells containing multiple nuclei appear in a short time after FLC treatment, we provide evidence that the heteroresistant colonies in the presence of FLC arise from uninucleate cells without involving multinuclear/multimeric stages. We found that fidelity of chromosome segregation in mitosis plays an important role in regulation of FLC heteroresistance frequency in C. neoformans. Although FLC-resistant colonies occurred at a very low frequency, we were able to modulate the frequency of heteroresistance by overexpressing SMC1, which encodes a protein containing an SMC domain in chromosome segregation. Using time-lapse microscopy, we captured the entire process of colony formation from a single cell in the presence of FLC. All the multinucleated cells formed within a few hours of FLC exposure failed to multiply after a few cell divisions, and the cells able to proliferate to form colonies were all uninucleate without exception. Furthermore, no nuclear fusion event or asymmetric survival between mother and daughter cells, a hallmark of chromosome nondisjunction in haploid organisms, was observed. Therefore, the mechanisms of aneuploidy formation in C. neoformans appear different from most common categories of aneuploid formation known for yeasts. IMPORTANCE The gold standard of cryptococcosis treatment consists of induction therapy with amphotericin B followed by lifelong maintenance therapy with fluconazole (FLC). However, prolonged exposure to FLC induces the emergence of clones heteroresistant to azoles. All the heteroresistant clones thus far analyzed have been shown to be aneuploids, but how the aneuploid is formed remains unclear. Aneuploidy in fungi and other eukaryotic cells is known to result most commonly from chromosome missegregation during cell division due to defects in any one of the multiple components and processes that are required for the formation of two genetically identical daughter cells. Although formation of multinucleated cells has been observed in cells exposed to FLC, evidence for the emergence of drug-resistant aneuploid populations directly from such cells has been lacking. We show the evidence that the aneuploid in fluconazole-heteroresistant clones of Cryptococcus neoformans is derived neither from multinucleated cells nor from chromosome missegregation.

ABSTRACT Invasive aspergillosis (IA) remains the primary cause of morbidity and mortality in chronic granulomatous disease (CGD) patients, often due to infection by Aspergillus species refractory to antifungals. This motivates the search for alternative treatments, including immunotherapy. We investigated the effect of exogenous type I interferon (IFN) activation on the outcome of IA caused by three Aspergillus species, A. fumigatus, A. nidulans, and A. tanneri, in CGD mice. The animals were treated with poly(I):poly(C) carboxymethyl cellulose poly-l-lysine (PICLC), a mimetic of double-stranded RNA, 24 h preinfection and postinfection. The survival rates and lung fungal burdens were markedly improved by PICLC immunotherapy in animals infected with any one of the three Aspergillus species. While protection from IA was remarkable, PICLC induction of type I IFN in the lungs surged 24 h posttreatment and returned to baseline levels by 48 h, suggesting that PICLC altered early events in protection against IA. Immunophenotyping of recruited leukocytes and histopathological examination of tissue sections showed that PICLC induced similar cellular infiltrates as those in untreated-infected mice, in both cases dominated by monocytic cells and neutrophils. However, the PICLC immunotherapy resulted in a marked earlier recruitment of the leukocytes. Unlike with conidia, infection with A. nidulans germlings reduced the protective effect of PICLC immunotherapy. Additionally, antibody depletion of neutrophils totally reversed the protection, suggesting that neutrophils are crucial for PICLC-mediated protection. Together, these data show that prophylactic PICLC immunotherapy prerecruits these cells, enabling them to attack the conidia and thus resulting in a profound protection from IA. IMPORTANCE Patients with chronic granulomatous disease (CGD) are highly susceptible to invasive aspergillosis (IA). While Aspergillus fumigatus is the most-studied Aspergillus species, CGD patients often suffer IA caused by A. nidulans, A. tanneri, and other rare species. These non-fumigatus Aspergillus species are more resistant to antifungal drugs and cause higher fatality rates than A. fumigatus. Therefore, alternative therapies are needed to protect CGD patients. We report an effective immunotherapy of mice infected with three Aspergillus species via PICLC dosing. While protection from IA was long lasting, PICLC induction of type I IFN surged but quickly returned to baseline levels, suggesting that PICLC was altering early events in IA. Interestingly, we found responding immune cells to be similar between PICLC-treated and untreated-infected mice. However, PICLC immunotherapy resulted in an earlier recruitment of the leukocytes and suppressed fungal growth. This study highlights the value of type I IFN induction in CGD patients.

ABSTRACT 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.

Lysophosphatidic acid (LPA) is a small ubiquitous lipid found in vertebrate and nonvertebrate organisms that mediates diverse biological actions and demonstrates medicinal relevance. LPA's functional roles are driven by extracellular signaling through at least six 7-transmembrane G protein-coupled receptors. These receptors are named LPA1–6 and signal through numerous effector pathways activated by heterotrimeric G proteins, including Gi/o, G12/13, Gq, and Gs. LPA receptor-mediated effects have been described in numerous cell types and model systems, both in vitro and in vivo, through gain- and loss-of-function studies. These studies have revealed physiological and pathophysiological influences on virtually every organ system and developmental stage of an organism. These include the nervous, cardiovascular, reproductive, and pulmonary systems. Disturbances in normal LPA signaling may contribute to a range of diseases, including neurodevelopmental and neuropsychiatric disorders, pain, cardiovascular disease, bone disorders, fibrosis, cancer, infertility, and obesity. These studies underscore the potential of LPA receptor subtypes and related signaling mechanisms to provide novel therapeutic targets.

Cryptococcus neoformans is the most common cause of fungal meningoencephalitis in AIDS patients. Depletion of CD4 cells, such as occurs during advanced AIDS, is known to be a critical risk factor for developing cryptococcosis. However, the role of HIV-induced innate inflammation in susceptibility to cryptococcosis has not been evaluated. Thus, we sought to determine the role of Type I IFN induction in host defense against cryptococci by treatment of C. neoformans (H99) infected mice with poly-ICLC (pICLC), a dsRNA virus mimic. Unexpectedly, pICLC treatment greatly extended survival of infected mice and reduced fungal burdens in the brain. Protection from cryptococcosis by pICLC-induced Type I IFN was mediated by MDA5 rather than TLR3. PICLC treatment induced a large, rapid and sustained influx of neutrophils and Ly6Chigh monocytes into the lung while suppressing the development of eosinophilia. The pICLC-mediated protection against H99 was CD4 T cell dependent and analysis of CD4 T cell polyfunctionality showed a reduction in IL-5 producing CD4 T cells, marginal increases in Th1 cells and dramatic increases in RORγt+ Th17 cells in pICLC treated mice. Moreover, the protective effect of pICLC against H99 was diminished in IFNγ KO mice and by IL-17A neutralization with blocking mAbs. Furthermore, pICLC treatment also significantly extended survival of C. gattii infected mice with reduced fungal loads in the lungs. These data demonstrate that induction of type I IFN dramatically improves host resistance against the etiologic agents of cryptococcosis by beneficial alterations in both innate and adaptive immune responses.

Invasive aspergillosis (IA) due to Aspergillus fumigatus is a major cause of mortality in immunocompromised patients. The discovery of highly fertile strains of A. fumigatus opened the possibility to merge classical and contemporary genetics to address key questions about this pathogen. The merger involves sexual recombination, selection of desired traits, and genomics to identify any associated loci. We constructed a highly fertile isogenic pair of A. fumigatus strains with opposite mating types and used them to investigate whether mating type is associated with virulence and to find the genetic loci involved in azole resistance. The pair was made isogenic by 9 successive backcross cycles of the foundational strain AFB62 (MAT1-1) with a highly fertile (MAT1-2) progeny. Genome sequencing showed that the F9 MAT1-2 progeny was essentially identical to the AFB62. The survival curves of animals infected with either strain in three different animal models showed no significant difference, suggesting that virulence in A. fumigatus was not associated with mating type. We then employed a relatively inexpensive, yet highly powerful strategy to identify genomic loci associated with azole resistance. We used traditional in vitro drug selection accompanied by classical sexual crosses of azole-sensitive with resistant isogenic strains. The offspring were plated under varying drug concentrations and pools of resulting colonies were analyzed by whole genome sequencing. We found that variants in 5 genes contributed to azole resistance, including mutations in erg11A (cyp51A), as well as multi-drug transporters, erg25, and in HMG-CoA reductase. The results demonstrated that with minimal investment into the sequencing of three pools from a cross of interest, the variation(s) that contribute any phenotype can be identified with nucleotide resolution. This approach can be applied to multiple areas of interest in A. fumigatus or other heterothallic pathogens, especially for virulence associated traits.

Previous reports have shown that individual neurons of the brain can display somatic genomic mosaicism of unknown function. In this study, we report altered genomic mosaicism in single, sporadic Alzheimer's disease (AD) neurons characterized by increases in DNA content and amyloid precursor protein (APP) gene copy number. AD cortical nuclei displayed large variability with average DNA content increases of ∼8% over non-diseased controls that were unrelated to trisomy 21. Two independent single-cell copy number analyses identified amplifications at the APP locus. The use of single-cell qPCR identified up to 12 copies of APP in sampled neurons. Peptide nucleic acid (PNA) probes targeting APP, combined with super-resolution microscopy detected primarily single fluorescent signals of variable intensity that paralleled single-cell qPCR analyses. These data identify somatic genomic changes in single neurons, affecting known and unknown loci, which are increased in sporadic AD, and further indicate functionality for genomic mosaicism in the CNS.

The ability to grow on media containing certain D-amino acids as a sole nitrogen source is widely utilized to differentiate Cryptococcus gattii from C. neoformans. We used the C. neoformans H99 and C. gattii R265 strains to dissect the mechanisms of D-amino acids utilization. We identified three putative D-amino acid oxidase (DAO) genes in both strains and showed that each DAO gene plays different roles in D-amino acid utilization in each strain. Deletion of DAO2 retarded growth of R265 on eleven D-amino acids suggesting its prominent role on D-amino acid assimilation in R265. All three R265 DAO genes contributed to growth on D-Asn and D-Asp. DAO3 was required for growth and detoxification of D-Glu by both R265 and H99. Although growth of H99 on most D-amino acids was poor, deletion of DAO1 or DAO3 further exacerbated it on four D-amino acids. Overexpression of DAO2 or DAO3 enabled H99 to grow robustly on several D-amino acids suggesting that expression levels of the native DAO genes in H99 were insufficient for growth on D-amino acids. Replacing the H99 DAO2 gene with a single copy of the R265 DAO2 gene also enabled its utilization of several D-amino acids. Results of gene and promoter swaps of the DAO2 genes suggested that enzymatic activity of Dao2 in H99 might be lower compared to the R265 strain. A reduction in virulence was only observed when all DAO genes were deleted in R265 but not in H99 indicating a pathobiologically exclusive role of the DAO genes in R265. These results suggest that C. neoformans and C. gattii divergently evolved in D-amino acid utilization influenced by their major ecological niches.

Cryptococcus neoformans encounters a low oxygen environment when it enters the human host. Here, we show that the conserved Ras1 (a small GTPase) and Cdc24 (the guanine nucleotide exchange factor for Cdc42) play an essential role in cryptococcal growth in hypoxia. Suppressor studies indicate that PTP3 functions epistatically downstream of both RAS1 and CDC24 in regulating hypoxic growth. Ptp3 shares sequence similarity to the family of phosphotyrosine-specific protein phosphatases and the ptp3Δ strain failed to grow in 1% O2. We demonstrate that RAS1, CDC24 and PTP3 function in parallel to regulate thermal tolerance but RAS1 and CDC24 function linearly in regulating hypoxic growth while CDC24 and PTP3 reside in compensatory pathways. The ras1Δ and cdc24Δ strains ceased to grow at 1% O2 and became enlarged but viable single cells. Actin polarization in these cells, however, was normal for up to eight hours after transferring to hypoxic conditions. Double deletions of the genes encoding Rho GTPase Cdc42 and Cdc420, but not of the genes encoding Rac1 and Rac2, caused a slight growth retardation in hypoxia. Furthermore, growth in hypoxia was not affected by the deletion of several central genes functioning in the pathways of cAMP, Hog1, or the two-component like phosphorylation system that are critical in the cryptococcal response to osmotic and genotoxic stresses. Interestingly, although deletion of HOG1 rescued the hypoxic growth defect of ras1Δ, cdc24Δ, and ptp3Δ, Hog1 was not hyperphosphorylated in these three mutants in hypoxic conditions. RNA sequencing analysis indicated that RAS1, CDC24 and PTP3 acted upon the expression of genes involved in ergosterol biosynthesis, chromosome organization, RNA processing and protein translation. Moreover, growth of the wild-type strain under low oxygen conditions was affected by sub-inhibitory concentrations of the compounds that inhibit these biological processes, demonstrating the importance of these biological processes in the cryptococcal hypoxia response.

ABSTRACT Urease in Cryptococcus neoformans plays an important role in fungal dissemination to the brain and causing meningoencephalitis. Although urea is not required for synthesis of apourease encoded by URE1, the available nitrogen source affected the expression of URE1 as well as the level of the enzyme activity. Activation of the apoenzyme requires three accessory proteins, Ure4, Ure6, and Ure7, which are homologs of the bacterial urease accessory proteins UreD, UreF, and UreG, respectively. A yeast two-hybrid assay showed positive interaction of Ure1 with the three accessory proteins encoded by URE4, URE6, and URE7. Metalloproteomic analysis of cryptococcal lysates using inductively coupled plasma mass spectrometry (ICP-MS) and a biochemical assay of urease activity showed that, as in many other organisms, urease is a metallocentric enzyme that requires nickel transported by Nic1 for its catalytic activity. The Ure7 accessory protein (bacterial UreG homolog) binds nickel likely via its conserved histidine-rich domain and appears to be responsible for the incorporation of Ni2+ into the apourease. Although the cryptococcal genome lacks the bacterial UreE homolog, Ure7 appears to combine the functions of bacterial UreE and UreG, thus making this pathogen more similar to that seen with the plant system. Brain invasion by the ure1, ure7, and nic1 mutant strains that lack urease activity was significantly less effective in a mouse model. This indicated that an activated urease and not the Ure1 protein was responsible for enhancement of brain invasion and that the factors required for urease activation in C. neoformans resemble those of plants more than those of bacteria. IMPORTANCE Cryptococcus neoformans is the major fungal agent of meningoencephalitis in humans. Although urease is an important factor for cryptococcal brain invasion, the enzyme activation system has not been studied. We show that urease is a nickel-requiring enzyme whose activity level is influenced by the type of available nitrogen source. C. neoformans contains all the bacterial urease accessory protein homologs and nickel transporters except UreE, a nickel chaperone. Cryptococcal Ure7 (a homolog of UreG) apparently functions as both the bacterial UreG and UreE in activating the Ure1 apoenzyme. The cryptococcal urease accessory proteins Ure4, Ure6, and Ure7 interacted with Ure1 in a yeast two-hybrid assay, and deletion of any one of these not only inactivated the enzyme but also reduced the efficacy of brain invasion. This is the first study showing a holistic picture of urease in fungi, clarifying that urease activity, and not Ure1 protein, contributes to pathogenesis in C. neoformans

We performed the first measurement of 30S+α resonant elastic scattering to experimentally examine the 30S(α, p) stellar reaction rate in type I x-ray bursts. These bursts are the most frequent thermonuclear explosions in the galaxy, resulting from thermonuclear runaway on the surface of accreting neutron star binaries. The 30S(α, p) reaction plays a critical role in burst models, yet very little is known about the compound nucleus 34Ar at these energies nor the reaction rate itself. We performed a measurement of alpha elastic scattering with a radioactive beam of 30S to experimentally probe the entrance channel. Utilizing a gaseous active target system and silicon detector array, we extracted the excitation function from 1.8 to 5.5 MeV near 160° in the center-of-mass frame. The experimental data were analyzed with an R-Matrix calculation, and we discovered several new resonances and extracted their quantum properties (resonance energy, width, spin, and parity). Finally, we calculated the narrow resonant thermonuclear reaction rate of 30S(α, p) for these new resonances.

Cryptococcal meningoencephalitis is the most common fungal disease in the central nervous system. The mechanisms by which Cryptococcus neoformans invades the brain are largely unknown. In this study, we found that C. neoformans-derived microvesicles (CnMVs) can enhance the traversal of the blood-brain barrier (BBB) by C. neoformans invitro. The immunofluorescence imaging demonstrates that CnMVs can fuse with human brain microvascular endothelial cells (HBMECs), the constituents of the BBB. This activity is presumably due to the ability of the CnMVs to activate HBMEC membrane rafts and induce cell fusogenic activity. CnMVs also enhanced C. neoformans infection of the brain, found in both infected brains and cerebrospinal fluid. In infected mouse brains, CnMVs are distributed inside and around C. neoformans-induced cystic lesions. GFAP (glial fibrillary acidic protein)-positive astrocytes were found surrounding the cystic lesions, overlapping with the 14-3-3-GFP (14-3-3-green fluorescence protein fusion) signals. Substantial changes could be observed in areas that have a high density of CnMV staining. This is the first demonstration that C. neoformans-derived microvesicles can facilitate cryptococcal traversal across the BBB and accumulate at lesion sites of C. neoformans-infected brains. Results of this study suggested that CnMVs play an important role in the pathogenesis of cryptococcal meningoencephalitis.

ABSTRACT The mating efficiency of 50 Aspergillus fumigatus isolates from both clinical and environmental sources was analyzed. Forty isolates completed the sexual cycle in 4 weeks with variable levels of fertility designated high, medium, or low. Two opposite-mating-type strains exhibiting the highest fertility, AFB62 (MAT1-1), isolated from a case of invasive aspergillosis, and AFIR928 (MAT1-2), isolated from the environment, were chosen as the supermater pair. Single cleistothecia obtained from a cross of the two strains harbored a minimum of 1 × 104 ascospores. The viability of ascospores increased with the age of the fruiting body, 17% at 4 weeks and reaching 95% at 20 weeks. AFB62 and AFIR928 were equally virulent in two different murine models, despite differences in their sources. High recombination frequencies were observed when the closely linked genes alb1 (AFUA_2G17600) and abr2 (AFUA_2G17530) were used as genetic markers. Comparative genome hybridization analyses revealed that only 86 genes (ca. 0.86% of the genome) are significantly diverged between AFB62 and AFIR928. The high fertility in a relatively short period, combined with a high degree of virulence and a high recombination frequency, demonstrates that the mating pair AFB62 and AFIR928 provides an excellent tool for genetic studies of A. fumigatus. IMPORTANCE Aspergillus fumigatus is a heterothallic fungal pathogen that causes life-threatening infections in immunocompromised hosts. Although heterothallism facilitates genetic study via recombinational analysis, previous work showed that a 6-month incubation period is required for the completion of sexual reproduction in this species. Such a long incubation period impedes progress in genetic research. To discover a highly fertile (supermater) pair that can complete the sexual cycle in a considerably shorter period, we screened 50 strains collected from various geographic regions for mating efficiency. We identified a highly virulent pair of supermaters that can be an invaluable tool for genetic study.

Human pluripotent stem cell (hPSC) lines have been considered to be homogeneously euploid. Here we report that normal hPSC--including induced pluripotent--lines are karyotypic mosaics of euploid cells intermixed with many cells showing non-clonal aneuploidies as identified by chromosome counting, spectral karyotyping (SKY) and fluorescent in situ hybridization (FISH) of interphase/non-mitotic cells. This mosaic aneuploidy resembles that observed in progenitor cells of the developing brain and preimplantation embryos, suggesting that it is a normal, rather than pathological, feature of stem cell lines. The karyotypic heterogeneity generated by mosaic aneuploidy may contribute to the reported functional and phenotypic heterogeneity of hPSCs lines, as well as their therapeutic efficacy and safety following transplantation.

Cryptococcus neoformans is a haploid environmental organism and the major cause of fungal meningoencephalitis in AIDS patients. Fluconazole (FLC), a triazole, is widely used for the maintenance therapy of cryptococcosis. Heteroresistance to FLC, an adaptive mode of azole resistance, was associated with FLC therapy failure cases but the mechanism underlying the resistance was unknown. We used comparative genome hybridization and quantitative real-time PCR in order to show that C. neoformans adapts to high concentrations of FLC by duplication of multiple chromosomes. Formation of disomic chromosomes in response to FLC stress was observed in both serotype A and D strains. Strains that adapted to FLC concentrations higher than their minimal inhibitory concentration (MIC) contained disomies of chromosome 1 and stepwise exposure to even higher drug concentrations induced additional duplications of several other specific chromosomes. The number of disomic chromosomes in each resistant strain directly correlated with the concentration of FLC tolerated by each strain. Upon removal of the drug pressure, strains that had adapted to high concentrations of FLC returned to their original level of susceptibility by initially losing the extra copy of chromosome 1 followed by loss of the extra copies of the remaining disomic chromosomes. The duplication of chromosome 1 was closely associated with two of its resident genes: ERG11, the target of FLC and AFR1, the major transporter of azoles in C. neoformans. This adaptive mechanism in C. neoformans may play an important role in FLC therapy failure of cryptococcosis leading to relapse during azole maintenance therapy.

The events required to initiate host defenses against invading pathogens involve complex signaling cascades comprised of numerous adaptor molecules, kinases, and transcriptional elements, ultimately leading to the production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha). How these signaling cascades are regulated, and the proteins and regulatory elements participating are still poorly understood.We report here the development a completely random short-hairpin RNA (shRNA) library coupled with a novel forward genetic screening strategy to identify inhibitors of Toll-like receptor (TLR) dependent proinflammatory responses. We developed a murine macrophage reporter cell line stably transfected with a construct expressing diphtheria toxin-A (DT-A) under the control of the TNF-alpha-promoter. Stimulation of the reporter cell line with the TLR ligand lipopolysaccharide (LPS) resulted in DT-A induced cell death, which could be prevented by the addition of an shRNA targeting the TLR adaptor molecule MyD88. Utilizing this cell line, we screened a completely random lentiviral short hairpin RNA (shRNA) library for sequences that inhibited TLR-mediated TNF-alpha production. Recovery of shRNA sequences from surviving cells led to the identification of unique shRNA sequences that significantly inhibited TLR4-dependent TNF-alpha gene expression. Furthermore, these shRNA sequences specifically blocked TLR2 but not TLR3-dependent TNF-alpha production.Thus, we describe the generation of novel tools to facilitate large-scale forward genetic screens in mammalian cells and the identification of potent shRNA inhibitors of TLR2 and TLR4- dependent proinflammatory responses.

Cryptococcus neoformans is an environmental fungal pathogen that requires atmospheric levels of oxygen for optimal growth. For the fungus to be able to establish an infection, it must adapt to the low oxygen concentrations in the host environment compared to its natural habitat. In order to investigate the oxygen sensing mechanism in C. neoformans, we screened T-DNA insertional mutants for hypoxia-mimetic cobalt chloride (CoCl(2))-sensitive mutants. All the CoCl(2)-sensitive mutants had a growth defect under low oxygen conditions at 37 degrees C. The majority of mutants are compromised in their mitochondrial function, which is reflected by their reduced rate of respiration. Some of the mutants are also defective in mitochondrial membrane permeability, suggesting the importance of an intact respiratory system for survival under both high concentrations of CoCl(2) as well as low oxygen conditions. In addition, the mutants tend to accumulate intracellular reactive oxygen species (ROS), and all mutants show sensitivity to various ROS generating chemicals. Gene expression analysis revealed the involvement of several pathways in response to cobalt chloride. Our findings indicate cobalt chloride sensitivity and/or sensitivity to low oxygen conditions are linked to mitochondrial function, sterol and iron homeostasis, ubiquitination, and the ability of cells to respond to ROS. These findings imply that multiple pathways are involved in oxygen sensing in C. neoformans.

BackgroundAspergillus fumigatus is the most common etiologic agent of invasive aspergillosis in immunocompromised patients. Several studies have addressed the mechanism involved in host defense but only few have investigated the pathogen's response to attack by the host cells. To our knowledge, this is the first study that investigates the genes differentially expressed in conidia vs hyphae of A. fumigatus in response to neutrophils from healthy donors as well as from those with chronic granulomatous disease (CGD) which are defective in the production of reactive oxygen species.Methodology/principal findingsTranscriptional profiles of conidia and hyphae exposed to neutrophils, either from normal donors or from CGD patients, were obtained by using the genome-wide microarray. Upon exposure to either normal or CGD neutrophils, 244 genes were up-regulated in conidia but not in hyphae. Several of these genes are involved in the degradation of fatty acids, peroxisome function and the glyoxylate cycle which suggests that conidia exposed to neutrophils reprogram their metabolism to adjust to the host environment. In addition, the mRNA levels of four genes encoding proteins putatively involved in iron/copper assimilation were found to be higher in conidia and hyphae exposed to normal neutrophils compared to those exposed to CGD neutrophils. Deletants in several of the differentially expressed genes showed phenotypes related to the proposed functions, i.e. deletants of genes involved in fatty acid catabolism showed defective growth on fatty acids and the deletants of iron/copper assimilation showed higher sensitivity to the oxidative agent menadione. None of these deletants, however, showed reduced resistance to neutrophil attack.ConclusionThis work reveals the complex response of the fungus to leukocytes, one of the major host factors involved in antifungal defense, and identifies fungal genes that may be involved in establishing or prolonging infections in humans.