Tubulin isoform composition tunes microtubule dynamics

Microtubules polymerize and depolymerize stochastically, a behavior essential for cell division, motility and differentiation. While many studies advanced our understanding of how microtubule-associated proteins tune microtubule dynamics in trans, we have yet to understand how tubulin genetic diversity regulates microtubule functions. The majority of in vitro dynamics studies are performed with tubulin purified from brain tissue. This preparation is not representative of tubulin found in many cell types. Here we report the 4.2Å cryo-EM structure and in vitro dynamics parameters of α1B/βI+βIVb microtubules assembled from tubulin purified from a human embryonic kidney cell line with isoform composition characteristic of fibroblasts and many immortalized cell lines. We find that these microtubules grow faster and transition to depolymerization less frequently compared to brain microtubules. Cryo-EM reveals that the dynamic ends of α1B/βI+βIVb microtubules are less tapered and that these tubulin heterodimers display lower curvatures. Interestingly, analysis of EB1 distributions at dynamic ends suggests no differences in GTP cap sizes. Lastly, we show that the addition of recombinant α1A/βIII tubulin, a neuronal isotype overexpressed in many tumors, proportionally tunes the dynamics of α1B/βI+βIVb microtubules. Our study is an important step towards understanding how tubulin isoform composition tunes microtubule dynamics

A population-based study of human immunodeficiency virus in south India reveals major differences from sentinel surveillance-based estimates

BACKGROUND: The human immunodeficiency virus (HIV) burden among adults in India is estimated officially by direct extrapolation of annual sentinel surveillance data from public-sector antenatal and sexually transmitted infection (STI) clinics and some high-risk groups. The validity of these extrapolations has not been systematically examined with a large sample population-based study. METHODS: We sampled 13838 people, 15–49 years old, from 66 rural and urban clusters using a stratified random method to represent adults in Guntur district in the south Indian state of Andhra Pradesh. We interviewed the sampled participants and obtained dried blood spots from them, and tested blood for HIV antibody, antigen and nucleic acid. We calculated the number of people with HIV in Guntur district based on these data, compared it with the estimate using the sentinel surveillance data and method, and analysed health services use data to understand the differences. RESULTS: In total, 12617 people (91.2% of the sampled group) gave a blood sample. Adjusted HIV prevalence was 1.72% (95% confidence interval 1.35–2.09%); men 1.74% (1.27–2.21%), women 1.70% (1.36–2.04%); rural 1.64% (1.10–2.18%), urban 1.89% (1.39–2.39%). HIV prevalence was 2.58% and 1.20% in people in the lower and upper halves of a standard of living index (SLI). Of women who had become pregnant during the past 2 years, 21.1% had used antenatal care in large public-sector hospitals participating in sentinel surveillance. There was an over-representation of the lowest SLI quartile (44.7%) in this group, and 3.61% HIV prevalence versus 1.08% in the remaining pregnant women. HIV prevalence was higher in that group even when women were matched for the same SLI half (lower half 4.39%, upper 2.63%) than in the latter (lower 1.06%, upper 1.05%), due to referral of HIV-positive/suspected women by private practitioners to public hospitals. The sentinel surveillance method (HIV prevalence: antenatal clinic 3%, STI clinic 22.8%, female sex workers 12.8%) led to an estimate of 112635 (4.38%) people with HIV, 15–49 years old, in Guntur district, which was 2.5 times the 45942 (1.79%) estimate based on our population-based study. CONCLUSION: The official method in India leads to a gross overestimation of the HIV burden in this district due to addition of substantial extra HIV estimates from STI clinics, the common practice of referral of HIV-positive/suspected people to public hospitals, and a preferential use of public hospitals by people in lower socioeconomic strata. India may be overestimating its HIV burden with the currently used official estimation method

Initial Commitment to Pre-Exposure Prophylaxis and Circumcision for HIV Prevention amongst Indian Truck Drivers

Studies of HIV prevention interventions such as pre-exposure prophylaxis (PREP) and circumcision in India are limited. The present study sought to investigate Indian truck-drivers initial commitment to PREP and circumcision utilizing the AIDS Risk Reduction Model. Ninety truck-drivers completed an in-depth qualitative interview and provided a blood sample for HIV and HSV-2 testing. Truck-drivers exhibited low levels of initial commitment towards PREP and even lower for circumcision. However, potential leverage points for increasing commitment were realized in fear of infecting family rather than self, self-perceptions of risk, and for PREP focusing on cultural beliefs towards medication and physicians. Cost was a major barrier to both HIV prevention interventions. Despite these barriers, our findings suggest that the ARRM may be useful in identifying several leverage points that may be used by peers, health care providers and public health field workers to enhance initial commitment to novel HIV prevention interventions in India

Structural determinants of microtubule minus end preference in CAMSAP CKK domains

CAMSAP/Patronins regulate microtubule minus-end dynamics. Their end specificity is mediated by their CKK domains, which we proposed recognise specific tubulin conformations found at minus ends. To critically test this idea, we compared the human CAMSAP1 CKK domain (HsCKK) with a CKK domain from Naegleria gruberi (NgCKK), which lacks minus-end specificity. Here we report near-atomic cryo-electron microscopy structures of HsCKK- and NgCKK-microtubule complexes, which show that these CKK domains share the same protein fold, bind at the intradimer interprotofilament tubulin junction, but exhibit different footprints on microtubules. NMR experiments show that both HsCKK and NgCKK are remarkably rigid. However, whereas NgCKK binding does not alter the microtubule architecture, HsCKK remodels its microtubule interaction site and changes the underlying polymer structure because the tubulin lattice conformation is not optimal for its binding. Thus, in contrast to many MAPs, the HsCKK domain can differentiate subtly specific tubulin conformations to enable microtubule minus-end recognition

Moving the Needle: Evidence of an Effective Study Strategy Intervention in a Community College Biology Course.

Many science, technology, engineering, and math (STEM) community college students do not complete their degree, and these students are more likely to be women or in historically excluded racial or ethnic groups. In introductory courses, low grades can trigger this exodus. Implementation of high-impact study strategies could lead to increased academic performance and retention. The examination of study strategies rarely occurs at the community college level, even though community colleges educate approximately half of all STEM students in the United States who earn a bachelor's degree. To fill this research gap, we studied students in two biology courses at a Hispanic-serving community college. Students were asked their most commonly used study strategies at the start and end of the semester. They were given a presentation on study skills toward the beginning of the semester and asked to self-assess their study strategies for each exam. We observed a significantly higher course grade for students who reported spacing their studying and creating drawings when controlling for demographic factors, and usage of these strategies increased by the end of the semester. We conclude that high-impact study strategies can be taught to students in community college biology courses and result in higher course performance

RT-PCR analysis of the putative co-operonic pairs <i>PE5-PPE4</i> and <i>PE15-PPE20</i>.

<p>Schematic representation of the genomic organization of the <i>PE5</i> (A) and <i>PE15</i> (C) loci in the <i>M.tb</i> genome showing the positions of the primers used in the study. (B) RT-PCR amplification products of the <i>PE5-PPE4</i> gene pair: Lane 1- (5F+5R), Lane 2- (5JF+4JR), Lane 3 - (4F+4R), Lanes 5, 6 and 7 correspond to -RT controls for these respective primer pairs, Lane 4–100 bp DNA ladder (D) RT-PCR amplification products of the <i>PE15-PPE20</i> gene pair: Lane 1- (15F+15R), Lane 2- (15JF+20JR), Lane 3 - (20F+20R); Lanes 5, 6 and 7 correspond to -RT controls for these respective primer pairs. Lane 4–100 bp DNA ladder.</p

Physiology of recombinant <i>M.smegmatis</i> strains expressing <i>PE5</i> and <i>PE15</i> in resting J774.1 macrophages.

<p>(A) CFU counts of <i>M.smegmatis</i> expressing empty vector (pMV261), <i>PE5</i> (PE5) and <i>PE15</i> (PE15) 24, 48 and 72 h post infection. (B) Input and T₀ (post-infection) CFU counts of infecting bacilli (± SEM). (C and D) Transcript levels of pro- and anti-inflammatory cytokines and <i>iNOS</i>, 24, 48 and 72 h post infection, p<0.05 for all data points. Error bars represent ± SEM from three biological replicates. **p<0.005, *p<0.05.</p

MAP kinase signalling and its correlation to IL-10 levels in infected THP-1 macrophages.

<p>Phosphorylation levels of p38 (A) and ERK1/2 (B) and their densitometric quantitation from resting THP-1 cells infected with <i>M.smegmatis</i> expressing pMV261, <i>PE5</i> and <i>PE15</i> 24 h post infection. Error bars represent ± SEM of three biological replicates. **p<0.005, *p<0.05. The western blots shown are representative of at least two biological replicates.</p