Use of TSAR, Thermal Shift Analysis in R, to identify Folic Acid as a Molecule that Interacts with HIV-1 Capsid.

Thermal shift assay (TSA) is a versatile biophysical technique for studying protein interactions. Here, we report a free, open-source software tool TSAR (Thermal Shift Analysis in R) to expedite and automate the analysis of thermal shift data derived either from individual experiments or large screens of chemical libraries. The TSAR package incorporates multiple, dynamic workflows to facilitate the analysis of TSA data and returns publication-ready graphics or processed results. Further, the package includes a graphic user interface (GUI) that enables easy use by non-programmers, aiming to simplify TSA analysis while diversifying visualization. To exemplify the utility of TSAR we screened a chemical library of vitamins to identify molecules that interact with the capsid protein (CA) of human immunodeficiency virus type 1 (HIV-1). Our data show that hexameric CA interacts with folic acid in vitro.

Deliberate self-poisoning in Australian adolescents is increased on school days.

Deliberate self-poisoning (DSP) in adolescents is increasing dramatically. Life at school is one of the most important life influences for this age group. This study aimed to investigate whether the frequency of DSP is higher during school term compared to holidays and whether this difference has become greater over time. This is an ecological study using Poisons Information Centre (PIC) data for all DSPs in 10-19 year olds from New South Wales, Tasmania and Australian Capital Territory that occurred between 2005 and 2018. For each call, the date of the poisoning was assigned as 'term' or 'holiday'. To control for population growth, calls were expressed as per 100,000 of the population per day. Multivariable Poisson regression was performed to investigate the combined impact of various predictors (state, sex, year, holiday/term, day of week, age) on call number. 26,432 calls were included in the analysis (73.6% female, 24.1% male and 2.3% unknown). Poisson regression showed significant effects for all predictors, with an increased likelihood of DSP during the school term compared with holidays and on Monday-Thursday compared with Saturday but only during the school term. DSP doubled between 2012 and 2017 and the disparity between DSP that occurs during term vs. holiday increased over that time frame. We conclude that some of the increase in DSP is likely due to school-specific stressors, hence the school environment is the ideal setting for self-harm prevention initiatives.

Sensitivity of blood and tissue diagnostics for gastrointestinal cytomegalovirus disease in solid organ transplant recipients.

BACKGROUND: Gastrointestinal (GI) cytomegalovirus (CMV) disease is the most common manifestation of tissue-invasive CMV infection in solid organ transplant (SOT) recipients, but the diagnostic yields of blood and tissue testing have not been systematically assessed in a large patient cohort. METHODS: We retrospectively identified consecutive SOT recipients with biopsy-confirmed GI CMV disease who had both tissue and blood (CMV polymerase chain reaction or antigenemia) diagnostic testing performed within 14 days of diagnosis. Descriptive statistics and logistic regression were used to assess the association between patient factors and viremia and the diagnostic yield of tests performed on biopsy specimens. RESULTS: A total of 101 patients (73% donor seropositive/recipient seronegative [D+/R-], 22% recipient seropositive [R+]) had GI CMV disease (58% upper, 22% lower, and 20% both) at a median of 185 days (range, 21-6345 days) post transplant. In multivariate analysis, R+ CMV serostatus (odds ratio [OR] 0.1 [0.0-0.4], P < 0.001) and diagnosis >6 months post transplant (OR 0.3 [0.1-0.9], P = 0.03) were each independently associated with absence of CMV viremia at time of diagnosis. In the subset of patients (n = 29) in whom both histopathology and viral culture were performed on biopsy specimens, 11 (39%) had CMV detected only by culture and had similar clinical characteristics and outcomes to those with positive histopathology (P > 0.05 for all comparisons). CONCLUSIONS: The sensitivity of viremia in SOT recipients with GI CMV disease is significantly lower in CMV-seropositive patients and in those >6 months post transplant. Addition of viral culture to endoscopic biopsy specimens significantly increases the diagnostic yield for GI CMV disease.

Restriction fragment mass polymorphism (RFMP) analysis based on MALDI-TOF mass spectrometry for detecting antiretroviral resistance in HIV-1 infected patients.

Viral genotype assessment is important for effective clinical management of HIV-1 infected patients, especially when access and/or adherence to antiretroviral treatment is reduced. In this study, we describe development of a matrix-assisted laser desorption/ionization-time of flight mass spectrometry-based viral genotyping assay, termed restriction fragment mass polymorphism (RFMP). This assay is suitable for sensitive, specific and high-throughput detection of multiple drug-resistant HIV-1 variants. One hundred serum samples from 60 HIV-1-infected patients previously exposed to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) were analysed for the presence of drug-resistant viruses using the RFMP and direct sequencing assays. Probit analysis predicted a detection limit of 223.02 copies/mL for the RFMP assay and 1268.11 copies/mL for the direct sequencing assays using HIV-1 RNA Positive Quality Control Series. The concordance rates between the RFMP and direct sequencing assays for the examined codons were 97% (K65R), 97% (T69Ins/D), 97% (L74VI), 97% (K103N), 96% (V106AM), 97% (Q151M), 97% (Y181C), 97% (M184VI) and 94% (T215YF) in the reverse transcriptase coding region, and 100% (D30N), 100% (M46I), 100% (G48V), 100% (I50V), 100% (I54LS), 99% (V82A), 99% (I84V) and 100% (L90M) in the protease coding region. Defined mixtures were consistently and accurately identified by RFMP at 5% relative concentration of mutant to wild-type virus while at 20% or greater by direct sequencing. The RFMP assay based on mass spectrometry proved to be sensitive, accurate and reliable for monitoring the emergence and early detection of HIV-1 genotypic variants that lead to drug resistance.

Preparation of biologically active single-chain variable antibody fragments that target the HIV-1 gp120 V3 loop.

KD-247 is a humanized monoclonal antibody that targets the third hypervariable (V3) loop of gp120. It can efficiently neutralize a broad panel of clade B, but not non-clade B, HIV-1 isolates. To overcome this limitation, we are seeking to prepare genetically-engineered single-chain variable fragments (scFvs) of KD-247 that will have broader neutralizing activity against both clade B and non-clade B HIV-1 isolates. Initial attempts of optimizing the expression of KD-247 scFv have resulted in the formation of insoluble protein. Therefore, we have established purification protocols to recover, purify, and refold the KD-247 scFv from inclusion bodies. The protocol involved step-wise refolding of denatured scFv by dilution, dialysis, and on-column nickel-affinity purification. Monomeric scFv was further purified by size-exclusion chromatography. Using far UV circular dichroism (CD) spectroscopy we confirmed the expected beta-sheet profile of the refolded KD-247 scFv. Importantly, the refolded KD-247 scFv showed neutralizing activity against replication-competent HIV-1 BaL and JR-FL Env pseudotyped HIV-1, at potency comparable to that of the native full-size KD-247 antibody. Ongoing studies focus on the application of this system in generating KD-247 scFv variants with the ability to neutralize clade B and non-clade B HIV-1 isolates.

Effect of translocation defective reverse transcriptase inhibitors on the activity of N348I, a connection subdomain drug resistant HIV-1 reverse transcriptase mutant.

4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a highly potent inhibitor of HIV-1 reverse transcriptase (RT). We have previously shown that its exceptional antiviral activity stems from a unique mechanism of action that is based primarily on blocking translocation of RT; therefore we named EFdA a Translocation Defective RT Inhibitor (TDRTI). The N348I mutation at the connection subdomain (CS) of HIV-1 RT confers clinically significant resistance to both nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs). In this study we tested EFdA-triphosphate (TP) together with a related compound, ENdA-TP (4'-ethynyl-2-amino-2'-deoxdyadenosine triphosphate) against HIV-1 RTs that carry clinically relevant drug resistance mutations: N348I, D67N/K70R/L210Q/T215F, D67N/K70R/L210Q/T215F/N348I, and A62V/V5I/F77L/F116Y/Q151M. We demonstrate that these enzymes remain susceptible to TDRTIs. Similar to WT RT, the N348I RT is inhibited by EFdA mainly at the point of incorporation through decreased translocation. In addition, the N348I substitution decreases the RNase H cleavage of DNA terminated with EFdA-MP (T/P(EFdA-MP)). Moreover, N348I RT unblocks EFdA-terminated primers with similar efficiency as the WT enzyme, and further enhances EFdA unblocking in the background of AZT-resistance mutations. This study provides biochemical insights into the mechanism of inhibition of N348I RT by TDRTIs and highlights the excellent efficacy of this class of inhibitors against WT and drug-resistant HIV-1 RTs.

The sugar ring conformation of 4'-ethynyl-2-fluoro-2'-deoxyadenosine and its recognition by the polymerase active site of HIV reverse transcriptase.

4' Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is the most potent inhibitor of HIV reverse transcriptase (RT). We have recently named EFdA a Translocation Defective RT Inhibitor (TDRTI) because after its incorporation in the nucleic acid it blocks DNA polymerization, primarily by preventing translocation of RT on the template/primer that has EFdA at the 3'-primer end (T/PEFdA). The sugar ring conformation of EFdA may also influence RT inhibition by a) affecting the binding of EFdA triphosphate (EFdATP) at the RT active site and/or b) by preventing proper positioning of the 3'-OH of EFdA in T/PEFdA that is required for efficient DNA synthesis. Specifically, the North (C2'-exo/C3'-endo), but not the South (C2'-endo/C3'-exo) nucleotide sugar ring conformation is required for efficient binding at the primer-binding and polymerase active sites of RT. In this study we use nuclear magnetic resonance (NMR) spectroscopy experiments to determine the sugar ring conformation of EFdA. We find that unlike adenosine nucleosides unsubstituted at the 4'-position, the sugar ring of EFdA is primarily in the North conformation. This difference in sugar ring puckering likely contributes to the more efficient incorporation of EFdATP by RT than dATP. In addition, it suggests that the 3'-OH of EFdA in T/PEFdA is not likely to prevent incorporation of additional nucleotides and thus it does not contribute to the mechanism of RT inhibition. This study provides the first insights into how structural attributes of EFdA affect its antiviral potency through interactions with its RT target.

Invasive Pseudomonas aeruginosa infections: high rate of recurrence and mortality after hematopoietic cell transplantation.

Limited data exist regarding the incidence and factors associated with outcome of invasive Pseudomonal infections in hematopoietic cell transplant (HCT). A retrospective analysis of cases of invasive Pseudomonas aeruginosa infection and factors associated with outcome was performed. P. aeruginosa invasive infection occurred in 95 of 5772 patients (1.65%) a median of 63 days after HCT (range 5-1435). Only 28% of infections occurred during periods of neutropenia (absolute neutrophil count<500 cells/mm(3)). Infection-attributable mortality during the initial episode of infection was 35.8%. Factors associated with initial mortality included the presence of a copathogen and high-dose steroid use. Ten (16.4%) of those who survived the initial infection experienced a recurrence of P. aeruginosa infection at a median of 9 days (range 3-17) after stopping antibiotics and 60% of those died as a result of recurrent infection a median of 1 day (range 1-7) after onset of recurrence. Grade 3-4 graft-versus-host disease was associated with a higher risk of recurrent infection. The risk of recurrence was not influenced by the presence of copathogens. Thus, invasive P. aeruginosa infections are associated with high recurrence rates and mortality in this immunocompromised population. Aggressive attempts to reduce immunosuppression and to treat copathogens may help during the initial infection.

Clinical and radiologic factors associated with pulmonary nodule etiology in organ transplant recipients.

Identifying clinical and radiographic factors that are associated with a specific etiology of pulmonary nodules (PNs) in solid-organ transplant (SOT) recipients might be helpful in guiding empiric therapy. Multivariable logistic regression was used to assess the relationship of clinical and radiographic variables to the etiology of PN in a retrospectively identified cohort of SOT recipients at a single transplant center. PNs in 55 SOT recipients (lung 15%, heart 22%, liver 42%, kidney 18% or kidney/pancreas 5%) were diagnosed at a mean of 1061 days post-transplant and were infectious in 31 of 55 (56%) (bacterial 22%, fungal 33%, viral 2%) and noninfectious in 24 of 55 (44%) [post-transplant lymphoproliferative disorder (PTLD) 25%, carcinoma 18%]. Radiographic 'consolidation' was independently associated with an infectious etiology (OR, 20.2, p < 0.01). Epstein-Barr virus seronegativity and lung transplant were each associated with PTLD (OR, 21.7, p < 0.01) and (OR, 36.6, p < 0.001), respectively. Diagnosis less than 90 days post-transplant was associated with Aspergillus infection (OR, 12.9, p = 0.007). Specific clinical and radiographic features are associated with specific etiologies of PNs in SOT recipients and might be useful for guiding empiric therapy while awaiting results of definitive diagnostic studies.

Body mass index, physical activity, and risk of renal cell carcinoma.

OBJECTIVE: To investigate the association between obesity and risk of renal cell carcinoma and to examine whether the association is modified by physical activity. SUBJECTS: A population-based case-control study of 406 patients with renal cell carcinoma and 2434 controls conducted in Iowa. METHODS: Information was collected on weight at the ages 20-29, 40-49, and 60-69 years, height, nonoccupational physical activity, diet, and other lifestyle factors. Renal cell carcinoma risk was estimated by odds ratios (ORs) and 95% confidence intervals (CIs), adjusting for age, total energy intake, and other confounding factors. RESULTS: Height and total energy intake were not associated with risk in either sex. In men, neither physical activity nor level of obesity in any period of life was significantly associated with risk. In women, lower physical activity was associated with higher risk (OR=2.5; 95% CI=1.2-5.2 comparing exercise <1 time/month to >1 time/day). Compared with women in the lowest quartile for BMI, the risks of renal cell carcinoma for women in the highest 10% of BMI in their 20s, 40s, and 60s were 1.4 (CI=0.6-3.1), 1.9 (CI=0.9-4.2), and 2.3 (CI=0.9-6.0), respectively. When analyses were limited to self-respondent data, the corresponding ORs were 2.9 (CI=1.2-7.4), 3.2 (CI=1.3-7.5), and 2.1 (CI=0.7-6.4), respectively. There was little evidence that physical activity modifies the association of BMI with renal cell carcinoma. CONCLUSION: Nonoccupational physical activity was inversely associated and obesity was positively associated with risk of renal cell carcinoma among women. The risk appeared to be greater for women in the highest 10% of BMI in their 40s. Our finding of little evidence of an interaction between physical activity and BMI requires confirmation.

What future direction should podiatric biomechanics take?

With researchers from many scientific and medical disciplines currently devoting countless hours trying to solve the mechanical mysteries of the foot, it is important that the proper direction for research be established. The author of this article believes that one of the most important directions in podiatric biomechanics will focus on the development of accurate models of the human foot and lower extremity. Accurate models will be useful because they provide relatively accurate predictions of the magnitudes of loading forces that occur in the structural component of the human foot and lower extremity during weight-bearing activities. From its beginnings in the early 1960s, podiatric biomechanics has developed from a fledgling collection of thoughts and observations from Merton Root and his colleagues to an internationally recognized clinical science with numerous researchers, past and present, contributing to its growing base of knowledge. With an emphasis on accurate modeling of the foot and lower extremity to allow better prediction of the internal forces that create pathologic conditions during weight-bearing activities, podiatric biomechanics will continue to provide valuable insight into many of the painful syndromes that plague children and adults of the world.

Subtalar joint axis location and rotational equilibrium theory of foot function.

A new theory of foot function based on the spatial location of the subtalar joint axis in relation to the weightbearing structures of the plantar foot is proposed. The theory relies on the concept of subtalar joint rotational equilibrium to explain how externally generated forces, such as ground reaction force, and internally generated forces, such as ligamentous and tendon tensile forces and joint compression forces, affect the mechanical behavior of the foot and lower extremity. The biomechanical effect of variations among individuals in the spatial location of the subtalar joint axis are explored, along with their clinical consequences, to offer an additional theory of foot function, which may improve on existing podiatric biomechanics theory.

Biomechanics of the normal and abnormal foot.

The foot is an engineering marvel that allows the body to perform many physical activities over a wide variety of terrain with remarkable efficiency. The functions of the foot and the lower extremity are biomechanically integrated; thus normal lower-extremity function requires normal foot function and vice versa. Because the subtalar joint is the main pedal joint allowing the triplanar translation of motion between the foot and lower extremity, normal subtalar joint function is critical to normal foot and lower-extremity function. This article provides an overview of the interrelationships between foot and lower-extremity function and mechanically based pathology of the foot and lower extremity, with an emphasis on the subtalar joint.

Distal ureteral stone in a duplicated system causing urinary retention.

Ureteral calculi and urinary retention are common problems encountered by the urologist. However, they rarely occur concomitantly. Herein, we describe a case of a 40-year-old female patient who developed urinary retention as a result of ureteral stone disease. Specifically, an ectopic upper pole ureter in a completely duplicated system contained a 2 x 6-cm ureteral stone, which emanated from the orifice, filling the urethra. The stone caused voiding difficulties to the extent that the patient had to manipulate the stone manually in order to void. The stone eventually resulted in urinary retention. Management was accomplished by cystoscopy and electrohydraulic lithotripsy of the stone. Chemical analysis revealed calcium phosphate and struvite as the principal components of the stone.

Intraluminal ultrasound-guided biopsy of the prostate: case report.

Patients who have undergone proctectomy can present a difficult diagnostic challenge for the urologist, as digital rectal examination and transrectal ultrasound scanning are not possible. Various methods have been tried to biopsy the prostate in patients without rectums and have proved to be limited in their usefulness. Intraluminal ultrasound employs a small high-frequency probe that creates an image from an intraluminal perspective. We describe a new role for this imaging method in which intraurethral intraluminal ultrasound scans can be used to guide perineal prostate biopsies in the patient without a rectum.

Fixed drug eruption to papaverine.

Papaverine has offered new options for therapy in erectile dysfunction. Various complications have been reported with papaverine, the prominent ones being priapism and liver function abnormalities. We present a previously unreported case of a fixed drug eruption caused by papaverine.