TTC12 Loss-of-Function Mutations Cause Primary Ciliary Dyskinesia and Unveil Distinct Dynein Assembly Mechanisms in Motile Cilia Versus Flagella.

Cilia and flagella are evolutionarily conserved organelles whose motility relies on the outer and inner dynein arm complexes (ODAs and IDAs). Defects in ODAs and IDAs result in primary ciliary dyskinesia (PCD), a disease characterized by recurrent airway infections and male infertility. PCD mutations in assembly factors have been shown to cause a combined ODA-IDA defect, affecting both cilia and flagella. We identified four loss-of-function mutations in TTC12, which encodes a cytoplasmic protein, in four independent families in which affected individuals displayed a peculiar PCD phenotype characterized by the absence of ODAs and IDAs in sperm flagella, contrasting with the absence of only IDAs in respiratory cilia. Analyses of both primary cells from individuals carrying TTC12 mutations and human differentiated airway cells invalidated for TTC12 by a CRISPR-Cas9 approach revealed an IDA defect restricted to a subset of single-headed IDAs that are different in flagella and cilia, whereas TTC12 depletion in the ciliate Paramecium tetraurelia recapitulated the sperm phenotype. Overall, our study, which identifies TTC12 as a gene involved in PCD, unveils distinct dynein assembly mechanisms in human motile cilia versus flagella.

Total scattering measurements at the Australian Synchrotron Powder Diffraction beamline: capabilities and limitations.

This study describes the capabilities and limitations of carrying out total scattering experiments on the Powder Diffraction (PD) beamline at the Australian Synchrotron, ANSTO. A maximum instrument momentum transfer of 19 Å-1 can be achieved if the data are collected at 21 keV. The results detail how the pair distribution function (PDF) is affected by Qmax, absorption and counting time duration at the PD beamline, and refined structural parameters exemplify how the PDF is affected by these parameters. There are considerations when performing total scattering experiments at the PD beamline, including (1) samples need to be stable during data collection, (2) highly absorbing samples with a µR > 1 always require dilution and (3) only correlation length differences >0.35 Šmay be resolved. A case study comparing the PDF atom-atom correlation lengths with EXAFS-derived radial distances of Ni and Pt nanocrystals is also presented, which shows good agreement between the two techniques. The results here can be used as a guide for researchers considering total scattering experiments at the PD beamline or similarly setup beamlines.

Specific Ion Effects at the Vapor-Formamide Interface: A Reverse Hofmeister Series in Ion Concentration Depth Profiles.

Employing neutral impact collision ion scattering spectroscopy (NICISS), we have directly measured the concentration depth profiles (CDPs) of various monovalent ions at the vapor-formamide interface. NICISS provides CDPs of individual ions by measuring the energy loss of neutral helium atoms backscattered from the solution interface. CDPs at the vapor-formamide interface of Cl-, Br-, I-, Na+, K+, and Cs+ are measured and compared to elucidate the interfacial specific ion trends. We report a reverse Hofmeister series in the presence of inorganic ions (anion and cation) at the vapor-formamide interface relative to the water-vapor interface, and the CDPs are found to be independent of the counterion for most ions studied. Thus, ions at the surface of formamide follow a "Hofmeister paradigm" where the counterion does not impact the ion series. These specific ion trends are complemented with surface tension and X-ray absorption near-edge structure (XANES) measurements on formamide electrolyte solutions.

Results and perinatal outcomes from 189 ICSI cycles of couples with asthenozoospermic men and flagellar defects assessed by transmission electron microscopy.

RESEARCH QUESTION: Do patients presenting with flagella ultrastructural defects as assessed by electron microscopy, and defined within three phenotypes (dysplasia of the fibrous sheath [DFS], primary flagellar dyskinesia [PFD] and non-specific flagellar abnormalities [NSFA]), have decreased chances of success in intracytoplasmic sperm injection (ICSI) or adverse obstetric and neonatal outcomes? DESIGN: Retrospective analysis of 189 ICSI cycles from 80 men with spermatozoa flagellum ultrastructural defects (DFS [n = 16]; PFD [n = 14]; NSFA [n = 50] compared with a control group (n = 97). Cycles were cumulatively analysed. All fresh and frozen embryo transfers resulting from each ICSI attempt were included. The effect of transmission electron microscopy (TEM) phenotype on the main ICSI outcomes was assessed by a multivariate logistic regression combined with a generalized linear mixed model to account for the non-independence of the observations. RESULTS: No predictive value of TEM phenotype was found on the main outcomes of ICSI, namely fertilization rates, pregnancy and delivery rates, and cumulative pregnancy and delivery rates. Cumulative pregnancy rates ranged from 29.0-43.3% in the different TEM phenotype subgroups compared with 36.8% in the control group. Cumulative live birth rates ranged from 24.6-36.7% compared with 31.4% in the control group. No increase was found in miscarriages, preterm births, low birth weights or birth abnormalities. CONCLUSIONS: Data on the cumulative chances of success in ICSI of patients with ultrastructural flagellar defects, a rare cause of male infertility often associated with an underlying genetic cause, are reassuring, as are obstetrical and neonatal outcomes in this population.

Mutations in DNAH17, Encoding a Sperm-Specific Axonemal Outer Dynein Arm Heavy Chain, Cause Isolated Male Infertility Due to Asthenozoospermia.

Motile cilia and sperm flagella share an evolutionarily conserved axonemal structure. Their structural and/or functional defects are associated with primary ciliary dyskinesia (PCD), a genetic disease characterized by chronic respiratory-tract infections and in which most males are infertile due to asthenozoospermia. Among the well-characterized axonemal protein complexes, the outer dynein arms (ODAs), through ATPase activity of their heavy chains (HCs), play a major role for cilia and flagella beating. However, the contribution of the different HCs (γ-type: DNAH5 and DNAH8 and ß-type: DNAH9, DNAH11, and DNAH17) in ODAs from both organelles is unknown. By analyzing five male individuals who consulted for isolated infertility and displayed a loss of ODAs in their sperm cells but not in their respiratory cells, we identified bi-allelic mutations in DNAH17. The isolated infertility phenotype prompted us to compare the protein composition of ODAs in the sperm and ciliary axonemes from control individuals. We show that DNAH17 and DNAH8, but not DNAH5, DNAH9, or DNAH11, colocalize with α-tubulin along the sperm axoneme, whereas the reverse picture is observed in respiratory cilia, thus explaining the phenotype restricted to sperm cells. We also demonstrate the loss of function associated with DNAH17 mutations in two unrelated individuals by performing immunoblot and immunofluorescence analyses on sperm cells; these analyses indicated the absence of DNAH17 and DNAH8, whereas DNAH2 and DNALI, two inner dynein arm components, were present. Overall, this study demonstrates that mutations in DNAH17 are responsible for isolated male infertility and provides information regarding ODA composition in human spermatozoa.

Going virtual during the COVID-19 pandemic: adaptation of a mixed-methods dietary behavior study within a community-based participatory research study of African-American adults at risk for cardiovascular disease.

BACKGROUND: Identifying mechanisms to maintain CBPR studies during an infectious disease pandemic is vital. The current paper describes the changes in methods and processes conducted within a CBPR mixed-methods study to a virtual setting during the novel coronavirus (COVID-19) pandemic. METHOD: The DC Community Organizing for Optimal Culinary Knowledge study with Heart (DC COOKS with Heart) was designed to assess the feasibility of a dietary behavior intervention among African-American adults that are at risk for cardiovascular disease (CVD). The study is under the umbrella of an ongoing CBPR study and community advisory board that facilitates community involvement in study design and promotes ongoing engagement with community members and leaders. The study population for D.C. COOKS with Heart consists of adult African-American individuals who live in two low-resource neighborhoods in Washington, D.C., which were impacted disproportionately by COVID. Eligible study participants who previously participated in the DC CHOC community-based studies were contacted to participate in Phase 1. The quantitative part of the mixed-methods included survey data collection. RESULTS: Due to the pandemic, the mode of data collection for surveys changed from self-administered face-to-face to internet-based. All virtual study procedures were conducted between March and April, 2021. Anticipated benefits of the virtual setting included participant safety during the pandemic, ease of logistics for participants. Anticipated challenges included administration of electronic devices to participants, research team training, and potential threats to established trust related to the privacy and confidentiality of participants. CONCLUSION: The transition to a virtual setting for study procedures in a mixed-methods study was conducted successfully in terms of recruitment, retention of participants, and training of research team members. The virtual transition required established and ongoing engagement through the community advisory board and CBPR practices, institutional support through virtual research policies, collaborations with information technology-based teams, and equipment administration for the study. TRIALS REGISTRATION: NCT04305431 . Registered on March 12, 2020.

Genetic analyses of a large cohort of infertile patients with globozoospermia, DPY19L2 still the main actor, GGN confirmed as a guest player.

Globozoospermia is a rare phenotype of primary male infertility inducing the production of round-headed spermatozoa without acrosome. Anomalies of DPY19L2 account for 50-70% of all cases and the entire deletion of the gene is by far the most frequent defect identified. Here, we present a large cohort of 69 patients with 20-100% of globozoospermia. Genetic analyses including multiplex ligation-dependent probe amplification, Sanger sequencing and whole-exome sequencing identified 25 subjects with a homozygous DPY19L2 deletion (36%) and 14 carrying other DPY19L2 defects (20%). Overall, 11 deleterious single-nucleotide variants were identified including eight novel and three already published mutations. Patients with a higher rate of round-headed spermatozoa were more often diagnosed and had a higher proportion of loss of function anomalies, highlighting a good genotype phenotype correlation. No gene defects were identified in patients carrying < 50% of globozoospermia while diagnosis efficiency rose to 77% for patients with > 50% of globozoospermia. In addition, results from whole-exome sequencing were scrutinized for 23 patients with a DPY19L2 negative diagnosis, searching for deleterious variants in the nine other genes described to be associated with globozoospermia in human (C2CD6, C7orf61, CCDC62, CCIN, DNAH17, GGN, PICK1, SPATA16, and ZPBP1). Only one homozygous novel truncating variant was identified in the GGN gene in one patient, confirming the association of GGN with globozoospermia. In view of these results, we propose a novel diagnostic strategy focusing on patients with at least 50% of globozoospermia and based on a classical qualitative PCR to detect DPY19L2 homozygous deletions. In the absence of the latter, we recommend to perform whole-exome sequencing to search for defects in DPY19L2 as well as in the other previously described candidate genes.

Neighborhood environment perceptions associate with depression levels and cardiovascular risk among middle-aged and older adults: Data from the Washington, DC cardiovascular health and needs assessment.

OBJECTIVES: Little is understood about associations between neighborhood characteristics and depression, a cardiovascular disease (CVD) risk factor, in diverse populations. We examined relationships between perceived/objective neighborhood characteristics, depression, and CVD markers within the Washington, DC CV Health/Needs Assessment, an evaluation among predominantly African-American (AA) adults in resource-limited DC communities. METHOD: Factor analysis of overall neighborhood environment perception (NEP) identified three NEP sub-scores:1) violence; 2) physical/social environment; 3) social cohesion (higher score = more favorable perception). Objective neighborhood characteristics were measured by geospatially-derived scores of walkability, transportation, and crime. Depression was defined by the revised Center for Epidemiologic Studies Depression Scale (CESD-R). We used linear-regression modeling to examine neighborhood measures and CESD-R associations. To investigate a subsequent connection with CVD risk, we examined relationships between CESD-R and CVD-associated cytokines in a population subset. RESULTS: Participants (N = 99; mean age = 59.06; 99% AA) had a mean CESD-R score = 5.8(SD = 8.88). In adjusted models, CESD-R scores decreased by 0.20 units (p = 0.01) for every overall NEP unit-increase. Perceived physical/social environment (ß = -0.34, p = 0.04) and social cohesion (ß = -0.82, p = 0.01) were related to CESD-R while perceived violence was not (ß = -0.28, p = 0.1). Of objective neighborhood environment measures (i.e. walk, transit, bike, personal crime, and property crime scores), only property crime score was associated with depression (ß = 4.99, p < 0.03). In population subset (n = 42), higher CESD-R associated with higher IL-1ß (ß = 21.25, p < 0.01) and IL-18 (ß = 0.006, p = 0.01). CONCLUSION: Favorable neighborhood perceptions are related to lower depressive symptoms in a predominantly AA cohort from Washington, DC resource-limited communities. Neighborhood perceptions appear to be strongly associated with depressive symptoms compared to objective characteristics. Increasing CESD-R scores were related to higher pro-inflammatory markers. Improving neighborhood perceptions may be beneficial to psychological well-being and CV health for urban minority residents.

Comparing Methods to Identify Wear-Time Intervals for Physical Activity With the Fitbit Charge 2.

There is no established method for processing data from commercially available physical activity trackers. This study aims to develop a standardized approach to defining valid wear time for use in future interventions and analyses. Sixteen African American women (mean age = 62.1 years and mean body mass index = 35.5 kg/m2) wore the Fitbit Charge 2 for 20 days. Method 1 defined a valid day as ≥10-hr wear time with heart rate data. Method 2 removed minutes without heart rate data, minutes with heart rate ≤ mean - 2 SDs below mean and ≤2 steps, and nighttime. Linear regression modeled steps per day per week change. Using Method 1 (n = 292 person-days), participants had 20.5 (SD = 4.3) hr wear time per day compared with 16.3 (SD = 2.2) hr using Method 2 (n = 282) (p < .0001). With Method 1, participants took 7,436 (SD = 3,543) steps per day compared with 7,298 (SD = 3,501) steps per day with Method 2 (p = .64). The proposed algorithm represents a novel approach to standardizing data generated by physical activity trackers. Future studies are needed to improve the accuracy of physical activity data sets.

Reduced Recombination and Capacitor-like Charge Buildup in an Organic Heterojunction.

Organic photovoltaic (OPV) efficiencies continue to rise, raising their prospects for solar energy conversion. However, researchers have long considered how to suppress the loss of free carriers by recombination-poor diffusion and significant Coulombic attraction can cause electrons and holes to encounter each other at interfaces close to where they were photogenerated. Using femtosecond transient spectroscopies, we report the nanosecond grow-in of a large transient Stark effect, caused by nanoscale electric fields of ∼487 kV/cm between photogenerated free carriers in the device active layer. We find that particular morphologies of the active layer lead to an energetic cascade for charge carriers, suppressing pathways to recombination, which is ∼2000 times less than predicted by Langevin theory. This in turn leads to the buildup of electric charge in donor and acceptor domains-away from the interface-resistant to bimolecular recombination. Interestingly, this signal is only experimentally obvious in thick films due to the different scaling of electroabsorption and photoinduced absorption signals in transient absorption spectroscopy. Rather than inhibiting device performance, we show that devices up to 600 nm thick maintain efficiencies of >8% because domains can afford much higher carrier densities. These observations suggest that with particular nanoscale morphologies the bulk heterojunction can go beyond its established role in charge photogeneration and can act as a capacitor, where adjacent free charges are held away from the interface and can be protected from bimolecular recombination.

Immune cell phenotyping in low blood volumes for assessment of cardiovascular disease risk, development, and progression: a pilot study.

BACKGROUND: Cardiovascular disease (CVD) is the leading cause of death in the world. Given the role of immune cells in atherosclerosis development and progression, effective methods for characterizing immune cell populations are needed, particularly among populations disproportionately at risk for CVD. RESULTS: By using a variety of antibodies combined in one staining protocol, we were able to identify granulocyte, lymphocyte, and monocyte sub-populations by CD-antigen expression from 500 µl of whole blood, enabling a more extensive comparison than what is possible with a complete blood count and differential (CBC). The flow cytometry panel was established and tested in a total of 29 healthy men and women. As a proof of principle, these 29 samples were split by their race/ethnicity: African-Americans (AA) (N = 14) and Caucasians (N = 15). We found in accordance with the literature that AA had fewer granulocytes and more lymphocytes when compared to Caucasians, though the proportion of total monocytes was similar in both groups. Several new differences between AA and Caucasians were noted that had not been previously described. For example, AA had a greater proportion of platelet adhesion on non-classical monocytes when compared to Caucasians, a cell-to-cell interaction described as crucially important in CVD. We also examined our flow panel in a clinical population of AA women with known CVD risk factors (N = 20). Several of the flow cytometry parameters that cannot be measured with the CBC displayed correlations with clinical CVD risk markers. For instance, Framingham Risk Score (FRS) calculated for each participant correlated with immune cell platelet aggregates (PA) (e.g. T cell PA ß = 0.59, p = 0.03 or non-classical monocyte PA ß = 0.54, p = 0.02) after adjustment for body mass index (BMI). CONCLUSION: A flow cytometry panel identified differences in granulocytes, monocytes, and lymphocytes between AA and Caucasians which may contribute to increased CVD risk in AA. Moreover, this flow panel identifies immune cell sub-populations and platelet aggregates associated with CVD risk. This flow cytometry panel may serve as an effective method for phenotyping immune cell populations involved in the development and progression of CVD.

Truncating Mutations in the Adhesion G Protein-Coupled Receptor G2 Gene ADGRG2 Cause an X-Linked Congenital Bilateral Absence of Vas Deferens.

In 80% of infertile men with obstructive azoospermia caused by a congenital bilateral absence of the vas deferens (CBAVD), mutations are identified in the cystic fibrosis transmembrane conductance regulator gene (CFTR). For the remaining 20%, the origin of the CBAVD is unknown. A large cohort of azoospermic men with CBAVD was retrospectively reassessed with more stringent selection criteria based on consistent clinical data, complete description of semen and reproductive excurrent ducts, extensive CFTR testing, and kidney ultrasound examination. To maximize the phenotypic prioritization, men with CBAVD and with unilateral renal agenesis were considered ineligible for the present study. We performed whole-exome sequencing on 12 CFTR-negative men with CBAVD and targeted sequencing on 14 additional individuals. We identified three protein-truncating hemizygous mutations, c.1545dupT (p.Glu516Ter), c.2845delT (p.Cys949AlafsTer81), and c.2002_2006delinsAGA (p.Leu668ArgfsTer21), in ADGRG2, encoding the epididymal- and efferent-ducts-specific adhesion G protein-coupled receptor G2, in four subjects, including two related individuals with X-linked transmission of their infertility. Previous studies have demonstrated that Adgrg2-knockout male mice develop obstructive infertility. Our study confirms the crucial role of ADGRG2 in human male fertility and brings new insight into congenital obstructive azoospermia pathogenesis. In men with CBAVD who are CFTR-negative, ADGRG2 testing could allow for appropriate genetic counseling with regard to the X-linked transmission of the molecular defect.

New insights into the morphological and hormonal characteristics of spermatogenic arrest.

The reported sperm retrieval rate (SRR) in patients with nonobstructive azoospermia (NOA) due to spermatogenic arrest (SA) is highly variable in the literature. This discrepancy could be explained by the heterogeneity of testicular tissues. Surprisingly, even though inhibin B levels reflect directly Sertoli cell function; no studies have evaluated this parameter in SA. We aimed to clarify the morphological and biological profile in 158 men with SA. From the total population, patients whose seminiferous tubules diameter was below 165 µm have higher SRR (46.9% vs. 27.4%, p < 0.05), lower inhibin levels and a higher frequency of nonuniform SA (71.9% vs. 38.7%, p < 0.001). On multivariate analysis, patients with late SA and a history of cryptorchidism were positively associated with successful sperm extraction. Patients with successful SRR and uniform SA exhibited inhibin levels twofold lower than those with failed TESE (45 pg/ml vs. 95 pg/ml, p < 0.05), whereas FSH levels were similar in the two groups. In this study, we showed for the first time that the mean diameter of the seminiferous tubules may be of value in the diagnosis of SA. Our results suggest that inhibin levels could be useful in the management of NOA with SA, along with FSH levels and testicular volume.

Defining the human sperm microtubulome: an integrated genomics approach.

Sperm motility notably depends on the structural integrity of the flagellum and the regulation of microtubule dynamics. Although researchers have started to use "omics" techniques to characterize the human sperm's molecular landscape, the constituents responsible for the assembly, organization, and dynamics of the flagellum microtubule have yet to be fully defined. In this study, we defined a core set of 116 gene products associated with the human sperm microtubulome (including products potentially involved in abnormal ciliary phenotypes and male infertility disorders). To this end, we designed and applied an integrated genomics workflow and combined relevant proteomics, transcriptomics, and interactomics datasets to reconstruct a dynamic interactome map. By further integrating phenotypic information, we defined a disease-interaction network; this enabled us to highlight a number of novel factors potentially associated with altered sperm motility and male fertility. Lastly, we experimentally validated the expression pattern of two candidate genes (CUL3 and DCDC2C) that had never previously been associated with male germline differentiation. Our analysis suggested that CUL3 and DCDC2C's products have important roles in the sperm flagellum. Taken as a whole, our results demonstrate that an integrated genomics strategy can highlight relevant molecular factors in specific sperm components. This approach could be easily extended by including other "omics" data (from asthenozoospermic men, for example) and identifying other critical proteins from the human sperm microtubulome.

A Green Route to Conjugated Polyelectrolyte Interlayers for High-Performance Solar Cells.

Synthesis of fluorene-based conjugated polyelectrolytes was achieved via Suzuki polycondensation in water and completely open to air. The polyelectrolytes were conveniently purified by dialysis and analysis of the materials showed properties expected for fluorene-based conjugated polyelectrolytes. The materials were then employed in solar cell devices as an interlayer in conjunction with ZnO. The double interlayer led to enhanced power conversion efficiency of 10.75 % and 15.1 % for polymer and perovskite solar cells, respectively.

Human Spermatozoa as a Model for Detecting Missing Proteins in the Context of the Chromosome-Centric Human Proteome Project.

The Chromosome-Centric Human Proteome Project (C-HPP) aims at cataloguing the proteins as gene products encoded by the human genome in a chromosome-centric manner. The existence of products of about 82% of the genes has been confirmed at the protein level. However, the number of so-called "missing proteins" remains significant. It was recently suggested that the expression of proteins that have been systematically missed might be restricted to particular organs or cell types, for example, the testis. Testicular function, and spermatogenesis in particular, is conditioned by the successive activation or repression of thousands of genes and proteins including numerous germ cell- and testis-specific products. Both the testis and postmeiotic germ cells are thus promising sites at which to search for missing proteins, and ejaculated spermatozoa are a potential source of proteins whose expression is restricted to the germ cell lineage. A trans-chromosome-based data analysis was performed to catalog missing proteins in total protein extracts from isolated human spermatozoa. We have identified and manually validated peptide matches to 89 missing proteins in human spermatozoa. In addition, we carefully validated three proteins that were scored as uncertain in the latest neXtProt release (09.19.2014). A focus was then given to the 12 missing proteins encoded on chromosomes 2 and 14, some of which may putatively play roles in ciliation and flagellum mechanistics. The expression pattern of C2orf57 and TEX37 was confirmed in the adult testis by immunohistochemistry. On the basis of transcript expression during human spermatogenesis, we further consider the potential for discovering additional missing proteins in the testicular postmeiotic germ cell lineage and in ejaculated spermatozoa. This project was conducted as part of the C-HPP initiatives on chromosomes 14 (France) and 2 (Switzerland). The mass spectrometry proteomics data have been deposited with the ProteomeXchange Consortium under the data set identifier PXD002367.

Loss-of-function mutations in LRRC6, a gene essential for proper axonemal assembly of inner and outer dynein arms, cause primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a group of autosomal-recessive disorders resulting from cilia and sperm-flagella defects, which lead to respiratory infections and male infertility. Most implicated genes encode structural proteins that participate in the composition of axonemal components, such as dynein arms (DAs), that are essential for ciliary and flagellar movements; they explain the pathology in fewer than half of the affected individuals. We undertook this study to further understand the pathogenesis of PCD due to the absence of both DAs. We identified, via homozygosity mapping, an early frameshift in LRRC6, a gene that encodes a leucine-rich-repeat (LRR)-containing protein. Subsequent analyses of this gene mainly expressed in testis and respiratory cells identified biallelic mutations in several independent individuals. The situs inversus observed in two of them supports a key role for LRRC6 in embryonic nodal cilia. Study of native LRRC6 in airway epithelial cells revealed that it localizes to the cytoplasm and within cilia, whereas it is absent from cells with loss-of-function mutations, in which DA protein markers are also missing. These results are consistent with the transmission-electron-microscopy data showing the absence of both DAs in cilia or flagella from individuals with LRRC6 mutations. In spite of structural and functional similarities between LRRC6 and DNAAF1, another LRR-containing protein involved in the same PCD phenotype, the two proteins are not redundant. The evolutionarily conserved LRRC6, therefore, emerges as an additional player in DA assembly, a process that is essential for proper axoneme building and that appears to be much more complex than was previously thought.

Evaluation of sperm nuclear integrity in patients with different percentages of decapitated sperm in ejaculates.

The decapitated sperm defect is a rare type of teratozoospermia responsible for male infertility. Spermatozoa from patients affected by this syndrome are used for intracytoplasmic sperm injection (ICSI) although little is known about their DNA integrity. This study evaluated sperm nuclear alterations in four patients and ten fertile men (control group). Sperm samples were examined by light, transmission electron and high-magnification contrast microscopy and analysed after terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling, aniline blue staining and fluorescence in-situ hybridization. Spermatozoa from patients presented varying degrees of decapitation, along with morphological and ultrastructural head abnormalities. Whereas the proportion of spermatozoa with fragmented DNA and numerical chromosome abnormalities was similar in patients 1-3 and controls, the percentage of spermatozoa with hypocondensed chromatin was higher in patients 1-3 than in fertile men. Patient 4 presented a distinct phenotype, with an increased proportion of flagellated spermatozoa with DNA strand breaks as well as increased aneuploidy and diploidy rates compared with controls and with patients 1-3. No successful pregnancy resulted from ICSI although embryos were obtained for three patients. The morphological defects and the nuclear alterations observed in spermatozoa of patients with the decapitated sperm syndrome may have contributed to ICSI failures.

Dissecting a complex chemical stress: chemogenomic profiling of plant hydrolysates.

The efficient production of biofuels from cellulosic feedstocks will require the efficient fermentation of the sugars in hydrolyzed plant material. Unfortunately, plant hydrolysates also contain many compounds that inhibit microbial growth and fermentation. We used DNA-barcoded mutant libraries to identify genes that are important for hydrolysate tolerance in both Zymomonas mobilis (44 genes) and Saccharomyces cerevisiae (99 genes). Overexpression of a Z. mobilis tolerance gene of unknown function (ZMO1875) improved its specific ethanol productivity 2.4-fold in the presence of miscanthus hydrolysate. However, a mixture of 37 hydrolysate-derived inhibitors was not sufficient to explain the fitness profile of plant hydrolysate. To deconstruct the fitness profile of hydrolysate, we profiled the 37 inhibitors against a library of Z. mobilis mutants and we modeled fitness in hydrolysate as a mixture of fitness in its components. By examining outliers in this model, we identified methylglyoxal as a previously unknown component of hydrolysate. Our work provides a general strategy to dissect how microbes respond to a complex chemical stress and should enable further engineering of hydrolysate tolerance.

Current drug use and lack of HIV virologic suppression: point-of-care urine drug screen versus self-report.

BACKGROUND: There have been inconsistent findings on the association between current drug use and HIV disease progression and virologic suppression. Drug use was often measured using self-report of historical use. Objective measurement of current drug use is preferred. METHODS: In this cross-sectional study, we assessed drug use through Computer-Assisted Self Interviews (CASI) and point-of-care urine drug screen (UDS) among 225 HIV-infected patients, and evaluated the association between current drug use and virologic suppression. RESULTS: About half (54%) of participants had a positive UDS, with a lower self-reported rate by CASI (42%) (Kappa score = 0.59). By UDS, 36.0% were positive for marijuana, 25.8% for cocaine, 7.6% for opiates, and 2.2% for methamphetamine or amphetamine. Factors associated with virologic suppression (plasma HIV RNA <50 copies/mL) were Caucasian race (P = 0.03), higher CD4 count (P < 0.01), current use of antiretroviral therapy (ART) (P < 0.01), and a negative UDS (P < 0.01). Among 178 current ART users, a positive UDS remained significantly associated with lower likelihood of virologic suppression (P = 0.04). CONCLUSIONS: UDS had good agreement with CASI in detecting frequently used drugs such as marijuana and cocaine. UDS at routine clinic visits may provide "real-time" prognostic information to optimize management.