Short-term observations of marine bacterial and viral communities: patterns,connections and resilience

Observation of short-term temporal variation in bacterial and viral communities is important for understanding patterns of aquatic microbial diversity. We collected surface seawater once daily for 38 consecutive days with seven more samples interspersed over 40 more days at one location ∼2 km from Santa Catalina Island, California. Bacterial communities were analyzed by automated ribosomal intergenic spacer analysis (ARISA) and viral communities were analyzed by terminal restriction fragment length polymorphism (TRFLP) of the conserved T4-like myoviral gene encoding the major capsid protein (g23). Common bacterial and viral taxa were consistently dominant, and relatively few displayed dramatic increases/decreases or ‘boom/bust'' patterns that might be expected from dynamic predator-prey interactions. Association network analysis showed most significant covariations (associations) occurred among bacterial taxa or among viral taxa and there were several modular (highly-interconnected) associations (P⩽0.005). Associations observed between bacteria and viruses (P⩽0.005) occurred with a median time lag of 2 days. Regression of all pairwise Bray-Curtis similarities between samples indicated a rate of bacterial community change that slows from 2.1%–0.18% per day over a week to 2 months; the rate stays around 0.4% per day for viruses. Our interpretation is that, over the scale of days, individual bacterial and viral OTUs can be dynamic and patterned; resulting in statistical associations regarded as potential ecological interactions. However, over the scale of weeks, average bacterial community variation is slower, suggesting that there is strong community-level ecological resilience, that is, a tendency to converge towards a ‘mean'' microbial community set by longer-term controlling factors.     

The AgI/II Family Adhesin AspA Is Required for Respiratory Infection by Streptococcus pyogenes

Streptococcus pyogenes (GAS) is a human pathogen that causes pharyngitis and invasive diseases such as toxic shock syndrome and sepsis. The upper respiratory tract is the primary reservoir from which GAS can infect new hosts and cause disease. The factors involved in colonisation are incompletely known however. Previous evidence in oral streptococci has shown that the AgI/II family proteins are involved. We hypothesized that the AspA member of this family might be involved in GAS colonization. We describe a novel mouse model of GAS colonization of the nasopharynx and lower respiratory tract to elucidate these interactions. We used two clinical M serotypes expressing AspA, and their aspA gene deletant isogenic mutants in experiments using adherence assays to respiratory epithelium, macrophage phagocytosis and neutrophil killing assays and in vivo models of respiratory tract colonisation and infection. We demonstrated the requirement for AspA in colonization of the respiratory tract. AspA mutants were cleared from the respiratory tract and were deficient in adherence to epithelial cells, and susceptible to phagocytosis. Expression of AspA in the surrogate host Lactococcus lactis protected bacteria from phagocytosis. Our results suggest that AspA has an essential role in respiratory infection, and may function as a novel anti-phagocytic factor.     

Histone Deacetylase Inhibitor Upregulates Peroxisomal Fatty Acid Oxidation and Inhibits Apoptotic Cell Death in Abcd1-Deficient Glial Cells

In X-ALD, mutation/deletion of ALD gene (ABCD1) and the resultant very long chain fatty acid (VLCFA) derangement has dramatically opposing effects in astrocytes and oligodendrocytes. While loss of Abcd1 in astrocytes produces a robust inflammatory response, the oligodendrocytes undergo cell death leading to demyelination in X-linked adrenoleukodystrophy (X-ALD). The mechanisms of these distinct pathways in the two cell types are not well understood. Here, we investigated the effects of Abcd1-knockdown and the subsequent alteration in VLCFA metabolism in human U87 astrocytes and rat B12 oligodendrocytes. Loss of Abcd1 inhibited peroxisomal β-oxidation activity and increased expression of VLCFA synthesizing enzymes, elongase of very long chain fatty acids (ELOVLs) (1 and 3) in both cell types. However, higher induction of ELOVL''s in Abcd1-deficient B12 oligodendrocytes than astrocytes suggests that ELOVL pathway may play a prominent role in oligodendrocytes in X-ALD. While astrocytes are able to maintain the cellular homeostasis of anti-apoptotic proteins, Abcd1-deletion in B12 oligodendrocytes downregulated the anti-apototic (Bcl-2 and Bcl-xL) and cell survival (phospho-Erk1/2) proteins, and upregulated the pro-apoptotic proteins (Bad, Bim, Bax and Bid) leading to cell loss. These observations provide insights into different cellular signaling mechanisms in response to Abcd1-deletion in two different cell types of CNS. The apoptotic responses were accompanied by activation of caspase-3 and caspase-9 suggesting the involvement of mitochondrial-caspase-9-dependent mechanism in Abcd1-deficient oligodendrocytes. Treatment with histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) corrected the VLCFA derangement both in vitro and in vivo, and inhibited the oligodendrocytes loss. These observations provide a proof-of principle that HDAC inhibitor SAHA may have a therapeutic potential for X-ALD.     

Direct Observation of Treatment Provided by a Family Member as Compared to Non-Family Member among Children with New Tuberculosis: A Pragmatic,Non-Inferiority,Cluster-Randomized Trial in Gujarat,India

Background

The World Health Organization recommends direct observation of treatment (DOT) to support patients with tuberculosis (TB) and to ensure treatment completion. As per national programme guidelines in India, a DOT provider can be anyone who is acceptable and accessible to the patient and accountable to the health system, except a family member. This poses challenges among children with TB who may be more comfortable receiving medicines from their parents or family members than from unfamiliar DOT providers. We conducted a non-inferiority trial to assess the effect of family DOT on treatment success rates among children with newly diagnosed TB registered for treatment during June–September 2012.

Methods

We randomly assigned all districts (n = 30) in Gujarat to the intervention (n = 15) or usual-practice group (n = 15). Adult family members in the intervention districts were given the choice to become their child’s DOT provider. DOT was provided by a non-family member in the usual-practice districts. Using routinely collected clinic-based TB treatment cards, we compared treatment success rates (cured and treatment completed) between the two groups and the non-inferiority limit was kept at 5%.

Results

Of 624 children with newly diagnosed TB, 359 (58%) were from intervention districts and 265 (42%) were from usual-practice districts. The two groups were similar with respect to baseline characteristics including age, sex, type of TB, and initial body weight. The treatment success rates were 344 (95.8%) and 247 (93.2%) (p = 0.11) among the intervention and usual-practice groups respectively.

Conclusion

DOT provided by a family member is not inferior to DOT provided by a non-family member among new TB cases in children and can attain international targets for treatment success.

Trial Registration

Clinical Trials Registry–India, National Institute of Medical Statistics (Indian Council of Medical Research) CTRI/2015/09/006229     

Sequential and compartmentalized action of Rabs,SNAREs, and MAL in the apical delivery of fusiform vesicles in urothelial umbrella cells

Uroplakins (UPs) are major differentiation products of urothelial umbrella cells and play important roles in forming the permeability barrier and in the expansion/stabilization of the apical membrane. Further, UPIa serves as a uropathogenic Escherichia coli receptor. Although it is understood that UPs are delivered to the apical membrane via fusiform vesicles (FVs), the mechanisms that regulate this exocytic pathway remain poorly understood. Immunomicroscopy of normal and mutant mouse urothelia show that the UP-delivering FVs contained Rab8/11 and Rab27b/Slac2-a, which mediate apical transport along actin filaments. Subsequently a Rab27b/Slp2-a complex mediated FV–membrane anchorage before SNARE-mediated and MAL-facilitated apical fusion. We also show that keratin 20 (K20), which forms a chicken-wire network ∼200 nm below the apical membrane and has hole sizes allowing FV passage, defines a subapical compartment containing FVs primed and strategically located for fusion. Finally, we show that Rab8/11 and Rab27b function in the same pathway, Rab27b knockout leads to uroplakin and Slp2-a destabilization, and Rab27b works upstream from MAL. These data support a unifying model in which UP cargoes are targeted for apical insertion via sequential interactions with Rabs and their effectors, SNAREs and MAL, and in which K20 plays a key role in regulating vesicular trafficking.     

Effects of an antiprogestin onapristone on the endometrium of bonnet monkeys: morphometric and ultrastructural studies

Our previous studies demonstrated the ability of low doses of antiprogestin ZK 98.299 (onapristone) to inhibit fertility in bonnet monkeys. In the present study cumulative effects of low doses of ZK 98.299 on the endometrial cytoarchitecture of bonnet monkeys were analyzed. Treatment with either the vehicle (n = 3) or onapristone at 2.5 mg (n = 4) or 5.0 mg (n = 3) was initiated on Day 5 of the first menstrual cycle and thereafter repeated every third day for four to seven consecutive cycles. The last treatment cycles were anovulatory in two animals treated with 2.5 mg and all animals treated with 5.0 mg. Endometrial biopsies were collected on Day 8 after the midcycle estradiol peak in ovulatory menstrual cycles and on Day 20 in anovulatory menstrual cycles during the last treatment cycle. Ultrathin sections of the fixed endometrium were stained with toluidine blue for morphometric analysis and uranyl acetate and lead citrate for ultrastructural analysis. The ZK 98.299-treated animals showed a dose-dependent endometrial atrophy as evident by a decrease in the height and diameter of the glands and early signs of compaction in the stroma. Ultrastructural analysis also revealed dose-dependent degenerative changes in the subcellular organelles such as the nucleus, mitochondria, endoplasmic reticulum, lysosomes, and Golgi apparatus. This suggests that long-term treatment with low doses of ZK 98.299 leads to the suppression of estrogen-dependent endometrial proliferation. However, this blockade operates independent of estradiol receptor (ER) and progesterone receptor (PR) concentrations as the expressions of these steroid receptors did not show any significant changes even after prolonged treatment. The study demonstrated an antiestrogenic effect of ZK 98.299 on endometrium after prolonged treatment in bonnet monkeys.     

Progesterone receptor as an indicator of sperm function

Expression of progesterone receptor (PR) localization on spermatozoa was determined in men with normal and abnormal spermiograms. Studies were also carried out to evaluate the potential of PR as a marker of sperm function. Progesterone receptor expression on spermatozoa from men with normozoospermia (n = 8), oligozoospermia (n = 7), asthenozoospermia (n = 8), oligoasthenozoospermia (n = 7), and teratozoospermia (n = 11) was analyzed using an immunocytochemical method with monoclonal antibodies against PR, and flow cytometry using a cell-impermeable fluorescein-tagged progesterone coupled to BSA complex (P-FITC-BSA). Both methods revealed significantly fewer (P < 0.05) PR-positive spermatozoa in men with oligozoospermia, asthenozoospermia, oligoasthenozoospermia, and teratozoospermia compared with men with normozoospermia, thereby suggesting that down-regulation of PR expression in spermatozoa may be one of the causes of male infertility. Spermatozoa from men with normozoospermia (n = 12), oligozoospermia (n = 12), asthenozoospermia (n = 12), oligoasthenozoospermia (n = 9), and teratozoospermia (n = 10) were exposed to low osmotic conditions in the hypoosmotic swelling (HOS) test and then analyzed for PR expression using P-FITC-BSA complex. A significantly higher percentage (P < 0.05) of spermatozoa with physiologically active plasma membrane (HOS+) lacked PR expression (HOS+PR-) in all categories of men with infertility, thereby suggesting that compared to the HOS test, PR expression is a better indicator of sperm function. Furthermore, PR expression in spermatozoa showed a strong (P < 0.05) positive correlation with their ability to undergo an in vitro acrosome reaction. This was observed in all study groups (i.e., normozoospermia, r = 0.8545; oligozoospermia, r = 0.8711; asthenozoospermia, r = 0.7645; oligoasthenozoospermia, r = 0.9003; and teratozoospermia, r = 0.8676). This suggests a potential role for PR in the events leading to the acrosome reaction in sperm.     

Functional Impairment of Myeloid Dendritic Cells during Advanced Stage of HIV-1 Infection: Role of Factors Regulating Cytokine Signaling

Introduction

Severely immunocompromised state during advanced stage of HIV-1 infection has been linked to functionally defective antigen presentation by dendritic cells (DCs). The molecular mechanisms behind DC impairment are still obscure. We investigated changes in DC function and association of key regulators of cytokine signaling during different stages of HIV-1 infection and following antiretroviral therapy (ART).

Methods

Phenotypic and functional characteristics of circulating myeloid DCs (mDCs) in 56 ART-naive patients (23 in early and 33 in advanced stage of disease), 36 on ART and 24 healthy controls were evaluated. Sixteen patients were studied longitudinally prior-to and 6 months after the start of ART. For functional studies, monocyte-derived DCs (Mo-DCs) were evaluated for endocytosis, allo-stimulation and cytokine secretion. The expression of suppressor of cytokine signaling (SOCS)-1 and other regulators of cytokine signaling was evaluated by real-time RT-PCR.

Results

The ability to respond to an antigenic stimulation was severely impaired in patients in advanced HIV-1 disease which showed partial recovery in the treated group. Mo-DCs from patients with advanced HIV-disease remained immature with low allo-stimulation and reduced cytokine secretion even after TLR-4 mediated stimulation ex-vivo. The cells had an increased expression of negative regulatory factors like SOCS-1, SOCS-3, SH2-containing phosphatase(SHP)-1 and a reduced expression of positive regulators like Janus kinase(JAK)2 and Nuclear factor kappa-light-chain-enhancer of activated B cells(NF-κB)1. A functional recovery after siRNA mediated silencing of SOCS-1 in these mo-DCs confirms the role of negative regulatory factors in functional impairment of these cells.

Conclusions

Functionally defective DCs in advanced stage of HIV-1 infection seems to be due to imbalanced state of negative and positive regulatory gene expression. Whether this is a cause or effect of increased viral replication at this stage of disease, needs further investigation. The information may be useful in design of novel therapeutic targets for better management of disease.