Kinetic Mechanism of DNA Translocation by the RSC Molecular Motor

ATP-dependent nucleosome repositioning by chromatin remodeling enzymes requires the translocation of these enzymes along the nucleosomal DNA. Using a fluorescence stopped-flow assay we monitored DNA translocation by a minimal RSC motor and through global analysis of these time courses we have determined that this motor has a macroscopic translocation rate of 2.9 bp/s with a step size of 1.24 bp. From the complementary quantitative analysis of the associated time courses of ATP consumption during DNA translocation we have determined that this motor has an efficiency of 3.0 ATP/bp, which is slightly less that the efficiency observed for several genetically related DNA helicases and which likely results from random pausing by the motor during translocation. Nevertheless, this motor is able to exert enough force during translocation to displace streptavidin from biotinylated DNA. Taken together these results are the necessary first step for quantifying both the role of DNA translocation in nucleosome repositioning by RSC and the efficiency at which RSC couples ATP binding and hydrolysis to nucleosome repositioning

Alterations in cathepsin H activity and protein patterns in human colorectal carcinomas

Our analyses of cathepsin H activity levels and protein forms in human colorectal cancers compared to matched control mucosa support the concept that altered proteinase expression patterns may reflect both cancer stage and site. Cathepsin H-specific activity was significantly increased in colorectal cancers compared to control mucosa (P = 0.003;n = 77). Highest specific activities and cancer/normal ratios (C/N) for activity were measured in Dukes' B and C stage carcinomas, cancers involved in local spread and invasion to lymph nodes. In contrast, cathepsin B and L activities analysed in the same paired extracts had been shown to be most frequently elevated in earlier stage carcinomas (Dukes' A and B), confirming that cathepsin H demonstrates a distinct pattern of expression during colorectal cancer progression. Although cathepsin H activities were most commonly elevated in Dukes' C cancers at all colon sites, both specific activity and C/N ratios were significantly higher for cancers of the left colon compared to other colon locations. A subset of 43 paired extracts analysed on Western blots also revealed consistent changes in cathepsin H protein forms in cancers. Normal mucosa typically showed a strong protein doublet at 31 and 29 kDa while cancers demonstrated decreased expression or total loss of the 31 kDa protein (90% of cases), equal or increased expression of the 29-kDa protein (67% of cases) and the new appearance or up-regulation of a cathepsin H band at 22 kDa (78% of cases). C/N ratios for cathepsin H enzyme activity correlated significantly with C/N ratios for the 29 kDa mature single-chain protein form (P< 0.001), with increased activity most commonly associated with elevated expression of 29-kDa cathepsin H but also with up-regulation of the 22-kDa band, suggesting a shift to more fully processed, mature active cathepsin H protein forms in cancers. Changes in cathepsin H expression were also detected by immunohistochemistry as elevated cathepsin H staining in tumour epithelial cells. © 2000 Cancer Research Campaig

Allosteric interactions of DNA and nucleotides with S. cerevisiae RSC

RSC, Remodel the Structure of Chromatin, is an essential chromatin remodeler of Saccharomyces cerevisiae that has been shown to have DNA translocase properties. We studied the DNA binding properties of a ‘trimeric minimal RSC’ (RSCt) of the RSC chromatin remodeling complex and the effect of nucleotides on this interaction using fluorescence anisotropy. RSCt binds to 20 bp fluorescein labeled double stranded DNA with a Kd of approximately 100 nM. The affinity of RSCt for DNA is reduced in the presence of AMP-PNP and ADP in a concentration dependent manner with the addition of AMP-PNP having the more pronounced effect. These differences in the magnitude at which the binding of ADP and AMP-PNP affect the affinity of DNA binding by RSCt suggests that the physical movement of the enzyme along DNA begins between the binding of ATP and its subsequent hydrolysis. Furthermore, the fact that the highest affinity for DNA binding by RSCt occurs in the absence of bound nucleotide offers a mechanistic explanation for the low apparent processivity of DNA translocation by the enzyme.This work was supported by National Institutes of Health Grant P20 RR017708 to C.J.F. and NIH GM60415 to B.R.C. Evan Rich was supported, in part, by an Undergraduate Research Award provided by the Kansas University Honors Program

A First Look at CQVID-19 Messages on WhatsApp in Pakistan

The worldwide spread of COVID-19 has prompted extensive online discussions, creating an `infodemic' on social media platforms such as WhatsApp and Twitter. However, the information shared on these platforms is prone to be unreliable and/or misleading. In this paper, we present the first analysis of COVID-19 discourse on public WhatsApp groups from Pakistan. Building on a large scale annotation of thousands of messages containing text and images, we identify the main categories of discussion. We focus on COVID-19 messages and understand the different types of images/text messages being propagated. By exploring user behavior related to COVID messages, we inspect how misinformation is spread. Finally, by quantifying the flow of information across WhatsApp and Twitter, we show how information spreads across platforms and how WhatsApp acts as a source for much of the information shared on Twitter

An exploration of the genetics of the mutant Huntingtin (mHtt) gene in a cohort of patients with chorea from different ethnic groups in sub-Saharan Africa

Background: Africans are underrepresented in Huntington\u27s disease (HD) research. A European ancestor was postulated to have introduced the mutant Huntingtin (mHtt) gene to the continent; however, recent work has shown the existence of a unique Htt haplotype in South-Africa specific to indigenous Africans. Objective: We aimed to investigate the CAG trinucleotide repeats expansion in the Htt gene in a geographically diverse cohort of patients with chorea and unaffected controls from sub-Saharan Africa. Methods: We evaluated 99 participants: 43 patients with chorea, 21 asymptomatic first-degree relatives of subjects with chorea, and 35 healthy controls for the presence of the mHtt. Participants were recruited from 5 African countries. Additional data were collected from patients positive for the mHtt gene; these included demographics, the presence of psychiatric and (or) cognitive symptoms, family history, spoken languages, and ethnic origin. Additionally, their pedigrees were examined to estimate the number of people at risk of developing HD and to trace back the earliest account of the disease in each region. Results: HD cases were identified in all countries. Overall, 53.4% of patients with chorea were carriers for the mHTT; median tract size was 45 CAG repeats. Of the asymptomatic relatives, 28.6% (6/21) were carriers for the mHTT; median tract size was 40 CAG. No homozygous carries were identified. Median CAG tract size in controls was 17 CAG repeats. Men and women were equally affected by HD. All patients with HD-bar three who were juvenile onset of \u3c21 years-were defined as adult onset (median age of onset was 40 years). HD transmission followed an autosomal dominant pattern in 84.2% (16/19) of HD families. In familial cases, maternal transmission was higher 52.6% (10/19) than paternal transmission 36.8% (7/19). The number of asymptomatic individuals at risk of developing HD was estimated at ten times more than the symptomatic patients. HD could be traced back to the early 1900s in most African sites. HD cases spread over seven ethnic groups belonging to two distinct linguistic lineages separated from each other approximately 54-16 kya ago: Nilo-Sahara and Niger-Congo. Conclusion: This is the first study examining HD in multiple sites in sub-Saharan Africa. We demonstrated that HD is found in multiple ethnic groups residing in five sub-Saharan African countries including the first genetically confirmed HD cases from Guinea and Kenya. The prevalence of HD in the African continent, its associated socio-economic impact, and genetic origins need further exploration and reappraisal

Testing for sexually transmitted infections in general practice: cross-sectional study

Background: Primary care is an important provider of sexual health care in England. We sought to explore the extent of testing for chlamydia and HIV in general practice and its relation to associated measures of sexual health in two contrasting geographical settings.Methods: We analysed chlamydia and HIV testing data from 64 general practices and one genitourinary medicine (GUM) clinic in Brent (from mid-2003 to mid-2006) and 143 general practices and two GUM clinics in Avon (2004). We examined associations between practice testing status, practice characteristics and hypothesised markers of population need (area level teenage conception rates and Index of Multiple Deprivation, IMD scores).Results: No HIV or chlamydia testing was done in 19% (12/64) of general practices in Brent, compared to 2.1% (3/143) in Avon. In Brent, the mean age of general practitioners (GPs) in Brent practices that tested for chlamydia or HIV was lower than in those that had not conducted testing. Practices where no HIV testing was done had slightly higher local teenage conception rates (median 23.5 vs. 17.4/1000 women aged 15-44, p = 0.07) and served more deprived areas (median IMD score 27.1 vs. 21.8, p = 0.05). Mean yearly chlamydia and HIV testing rates, in practices that did test were 33.2 and 0.6 (per 1000 patients aged 15-44 years) in Brent, and 34.1 and 10.3 in Avon, respectively. In Brent practices only 20% of chlamydia tests were conducted in patients aged under 25 years, compared with 39% in Avon.Conclusions: There are substantial geographical differences in the intensity of chlamydia and HIV testing in general practice. Interventions to facilitate sexually transmitted infection and HIV testing in general practice are needed to improve access to effective sexual health care. The use of routinely-collected laboratory, practice-level and demographic data for monitoring sexual health service provision and informing service planning should be more widely evaluated

The Macroscopic Rate of Nucleic Acid Translocation by Hepatitis C virus Helicase NS3h is Dependent on Both the Sugar and Base Moieties

The NS3 helicase (NS3h) of hepatitis C virus (HCV) is a 3′ to 5′ SF2 RNA and DNA helicase that is essential for the replication of HCV. We have examined the kinetic mechanism of translocation of NS3h along single-stranded nucleic acid with bases rU, dU and dT and have found that the macroscopic rate of translocation is dependent upon both the base and sugar moieties of the nucleic acid, with approximate macroscopic translocation rates of 3 nt/s (oligo-dT), 35 nt/s (oligo-dU), and 42 nt/s (oligo-rU), respectively. We found a strong correlation between the macroscopic translocation rates and the binding affinity of the translocating NS3h protein to the respective substrates such that weaker affinity corresponded to faster translocation. The values of K0.5 for NS3h translocation at a saturating ATP concentration are: (3.3 ± 0.4) μM nucleotide (poly-dT), (27 ± 2) μM nucleotide (poly-dU), and (36 ± 2) μM nucleotide (poly-rU). Furthermore, the results of isothermal titration of NS3h with these oligonucleotides suggest that differences in TΔS° are the principal source of the differences in the affinity of NS3h binding to these substrates. Interestingly, despite the differences in macroscopic translocation rates and binding affinities, the ATP coupling stoichiometry for NS3h translocation was identical for all three substrates, ~0.5 ATP molecules consumed per nucleotide translocated. This similar periodicity of ATP consumption implies a similar mechanism for NS3h translocation along RNA and DNA substrates

Thermomechanical modelling of laser surface glazing for H13 tool steel

A two-dimensional thermomechanical finite element (FE) model of laser surface glazing (LSG) has been developed for H13 tool steel. The direct coupling technique of ANSYS 17.2 (APDL) has been utilised to solve the transient thermomechanical process. A H13 tool steel cylindrical cross-section has been modelled for laser power 200 W and 300 W at constant 0.2 mm beam width and 0.15 ms residence time. The model can predict temperature distribution, stress–strain increments in elastic and plastic region with time and space. The crack formation tendency also can be assumed by analysing the von Mises stress in the heat-concentrated zone. Isotropic and kinematic hardening models have been applied separately to predict the after-yield phenomena. At 200 W laser power, the peak surface temperature achieved is 1520 K which is below the melting point (1727 K) of H13 tool steel. For laser power 300 W, the peak surface temperature is 2523 K. Tensile residual stresses on surface have been found after cooling, which are in agreement with literature. Isotropic model shows higher residual stress that increases with laser power. Conversely, kinematic model gives lower residual stress which decreases with laser power. Therefore, both plasticity models could work in LSG for H13 tool steel

6G Opportunities Arising from Internet of Things Use Cases: A Review Paper

The race for the 6th generation of wireless networks (6G) has begun. Researchers around the world have started to explore the best solutions for the challenges that the previous generations have experienced. To provide the readers with a clear map of the current developments, several review papers shared their vision and critically evaluated the state of the art. However, most of the work is based on general observations and the big picture vision, and lack the practical implementation challenges of the Internet of Things (IoT) use cases. This paper takes a novel approach in the review, as we present a sample of IoT use cases that are representative of a wide variety of its implementations. The chosen use cases are from the most research-active sectors that can benefit from 6G and its enabling technologies. These sectors are healthcare, smart grid, transport, and Industry 4.0. Additionally, we identified some of the practical challenges and the lessons learned in the implementation of these use cases. The review highlights the cases’ main requirements and how they overlap with the key drivers for the future generation of wireless networks