Limited secretory-IgA response in cervicovaginal secretions from HIV-1 infected, but not high risk seronegative women: lack of correlation to genital viral shedding

The presence and antigen specificity of IgG and secretory-IgA (s-IgA) to HIV-1 were evaluated in cervicovaginal lavages (CVL) from 26 infected and 10 high-risk seronegative women. All the seropositive women had detectable IgG recognizing several viral antigens, while a smaller percentage of women demonstrated s-IgA to the virus. In addition, s-IgA were of limited specificity and provided weak reactivities on Immunoblot bands; an almost constant absence of s-IgA to gp120 was also observed. Neither the presence nor the specificity of either IgG or s-IgA to the virus in CVL prevented the shedding of HIV-1 in this body fluid; in fact, viral RNA was detected in all the women studied and the amounts of viral shedding was unrelated to the genital antibody response. On the other hand, none of the high-risk seronegative women had detectable antibodies to HIV-1 in CVL of either the IgG or s-IgA isotype. Our results a) confirm an impairment of mucosal antibody response during HIV-1 infection and suggest that mucosal immunity is not able to prevent viral shedding in the female genital tract and thus cannot modulate the infectivity of genital secretions; aa) do not provide evidence for a mucosal "memory/protective" antibody response in the genital tract of high-risk seronegative women.     

Tyrosine phosphorylation of Eps15 is required for ligand-regulated, but not constitutive, endocytosis

Membrane receptors are internalized either constitutively or upon ligand engagement. Whereas there is evidence for differential regulation of the two processes, little is known about the molecular machinery involved. Previous studies have shown that an unidentified kinase substrate is required for endocytosis of the epidermal growth factor receptor (EGFR), the prototypical ligand-inducible receptor, but not of the transferrin receptor (TfR), the prototypical constitutively internalized receptor. Eps15, an endocytic protein that is tyrosine phosphorylated by EGFR, is a candidate for such a function. Here, we show that tyrosine phosphorylation of Eps15 is necessary for internalization of the EGFR, but not of the TfR. We mapped Tyr 850 as the major in vivo tyrosine phosphorylation site of Eps15. A phosphorylation-negative mutant of Eps15 acted as a dominant negative on the internalization of the EGFR, but not of the TfR. A phosphopeptide, corresponding to the phosphorylated sequence of Eps15, inhibited EGFR endocytosis, suggesting that phosphotyrosine in Eps15 serves as a docking site for a phosphotyrosine binding protein. Thus, tyrosine phosphorylation of Eps15 represents the first molecular determinant, other than those contained in the receptors themselves, which is involved in the differential regulation of constitutive vs. regulated endocytosis.     

Numb is an endocytic protein

Numb is a protein that in Drosophila determines cell fate as a result of its asymmetric partitioning at mitosis. The function of Numb has been linked to its ability to bind and to biologically antagonize Notch, a membrane receptor that also specifies cell fate. The biochemical mechanisms underlying the action of Numb, however, are still largely unknown. The wide pattern of expression of Numb suggests a general function in cellular homeostasis that could be additional to, or part of, its action in fate determination. Such a function could be endocytosis, as suggested by the interaction of Numb with Eps15, a component of the endocytic machinery. Here, we demonstrate that Numb is an endocytic protein. We found that Numb localizes to endocytic organelles and is cotrafficked with internalizing receptors. Moreover, it associates with the appendage domain of alpha adaptin, a subunit of AP2, a major component of clathrin-coated pits. Finally, fragments of Numb act as dominant negatives on both constitutive and ligand-regulated receptor-mediated internalization, suggesting a general role for Numb in the endocytic process.     

Free D- and L-amino acids in ventricular cerebrospinal fluid from Alzheimer and normal subjects

Summary Free D-Ser, D-Asp and total D-amino acids were significantly higher (p < 0.05) in Alzheimer (AD) ventricular CSF than in normal CSF. There was no significant difference in the total L-amino acids between AD and normal CSF, but L-Gln and L-His were significantly higher (p < 0.05) in ADCSF. The higher concentrations of these D- and L-amino acids in AD ventricular CSF could reflect the degenerative process that occurs in Alzheimer's brain since ventricular CSF is the repository of amino acids from the brain.     

Electronic Cigarettes Efficacy and Safety at 12 Months: Cohort Study

Objective

To evaluate the safety and efficacy as a tool of smoking cessation of electronic cigarettes (e-cigarettes), directly comparing users of e-cigarettes only, smokers of tobacco cigarettes only, and smokers of both.

Design

Prospective cohort study. Final results are expected in 2019, but given the urgency of data to support policies on electronic smoking, we report the results of the 12-month follow-up.

Data Sources

Direct contact and structured questionnaires by phone or via internet.

Methods

Adults (30–75 years) were included if they were smokers of ≥1 tobacco cigarette/day (tobacco smokers), users of any type of e-cigarettes, inhaling ≥50 puffs weekly (e-smokers), or smokers of both tobacco and e-cigarettes (dual smokers). Carbon monoxide levels were tested in a sample of those declaring tobacco smoking abstinence.

Main Outcome Measures

Sustained smoking abstinence from tobacco smoking at 12 months, reduction in the number of tobacco cigarettes smoked daily.

Data Synthesis

We used linear and logistic regression, with region as cluster unit.

Results

Follow-up data were available for 236 e-smokers, 491 tobacco smokers, and 232 dual smokers (overall response rate 70.8%). All e-smokers were tobacco ex-smokers. At 12 months, 61.9% of the e-smokers were still abstinent from tobacco smoking; 20.6% of the tobacco smokers and 22.0% of the dual smokers achieved tobacco abstinence. Adjusting for potential confounders, tobacco smoking abstinence or cessation remained significantly more likely among e-smokers (adjusted OR 5.19; 95% CI: 3.35–8.02), whereas adding e-cigarettes to tobacco smoking did not enhance the likelihood of quitting tobacco and did not reduce tobacco cigarette consumption. E-smokers showed a minimal but significantly higher increase in self-rated health than other smokers. Non significant differences were found in self-reported serious adverse events (eleven overall).

Conclusions

Adding e-cigarettes to tobacco smoking did not facilitate smoking cessation or reduction. If e-cigarette safety will be confirmed, however, the use of e-cigarettes alone may facilitate quitters remaining so.

Registration Number

NCT01785537.     

Understanding biological dynamics: following cells and molecules to track functions and mechanisms

The dissection of the molecular circuitries at the base of cell life and the identification of their abnormal transformation during carcinogenesis rely on the characterization of biological phenotypes generated by targeted overexpression or deletion of gene products through genetic manipulation. Fluorescence microscopy provides a wide variety of tools to monitor cell life with minimal perturbations. The observation of living cells requires the selection of a correct balance between temporal, spatial and “statistical” resolution according to the process to be analyzed. In the following paper ad hoc developed optical tools for dynamical tracking from cellular to molecular resolution will be presented. Particular emphasis will be devoted to discuss how to exploit light–matter interaction to selectively target specific molecular species, understanding the relationships between their intracellular compartmentalization and function.     

Development of a repressible mycobacterial promoter system based on two transcriptional repressors

Tightly regulated gene expression systems represent invaluable tools for studying gene function and for the validation of drug targets in bacteria. While several regulated bacterial promoters have been characterized, few of them have been successfully used in mycobacteria. In this article we describe the development of a novel repressible promoter system effective in both fast- and slow-growing mycobacteria based on two chromosomally encoded repressors, dependent on tetracycline (TetR) and pristinamycin (Pip), respectively. This uniqueness results in high versatility and stringency. Using this method we were able to obtain an ftsZ conditional mutant in Mycobacterium smegmatis and a fadD32 conditional mutant in Mycobacterium tuberculosis, confirming their essentiality for bacterial growth in vitro. This repressible promoter system could also be exploited to regulate gene expression during M. tuberculosis intracellular growth.     

Antibiotic resistance in long-term care facilities

Long-term-care facilities (LTCFs) comprise a heterogeneous group of institutions that provide a wide variety of services to diverse groups of patients, most of whom are elderly. Infections are common in LTCFs and these are complicated by antimicrobial-resistant pathogens. The residents in LTCFs have a high frequency of colonization with antimicrobial-resistant organisms, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, penicillin-resistant pneumococci, extended spectrum P-lactamase-producing gram-negative organisms, and fluoroquinolone-resistant gram-negative organisms. Although several control programs have been published, up to now there still is a long way to go in this area of health care. This review will briefly touch upon the clinical relevance of antimicrobial resistance in LTCFs.