The value of neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio, red cell distribution width, and their combination in predicting acute pancreatitis severity.

OBJECTIVE: Acute pancreatitis is one of the most common causes of acute abdominal pain requiring hospitalization worldwide. The neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and red blood cell distribution width (RDW) are novel inflammatory markers that have been investigated in various diseases associated with an inflammatory response, achieving many positive results. Evaluating the NLR, PLR, RDW, and their combination to predict acute pancreatitis severity can help clinicians have an appropriate initial treatment strategy. PATIENTS AND METHODS: This prospective cohort study enrolled 131 patients diagnosed with acute pancreatitis at Gia Dinh Hospital, Ho Chi Minh City, between December 2021 and August 2022. Patients with the following features were excluded from our study: age < 18 years old, time from symptom onset to admission of > 72 hours; patients with autoimmune disease, decompensated cirrhosis, active tuberculosis, heart failure (New York Heart Association class 4), end-stage renal failure, pregnancy, active severe acute respiratory syndrome coronavirus 2 infection, and chronic pancreatitis. RESULTS: There were 21 severe acute pancreatitis (SAP) cases (16%). The area under the receiver operating characteristic curve for predicting SAP was 0.82 for NLR, 0.72 for PLR, and 0.73 for RDW. When the cutoffs of 13.5 for NLR, 202.7 for PLR, and 13.1% for the RDW were used, the negative predictive values in predicting SAP were 93.1%, 91.9%, and 98.8%, respectively. This finding demonstrates the value of inflammatory markers in predicting SAP. The combination of these markers did not show an advantage in predicting SAP compared to the single markers. CONCLUSIONS: High NLR, PLR, and RDW are associated with SAP. These indices are good indicators for predicting SAP. In our study, the combination of inflammatory markers did not improve SAP prediction compared to the individual markers.

A quinary WTaCrVHf nanocrystalline refractory high-entropy alloy withholding extreme irradiation environments.

In the quest of new materials that can withstand severe irradiation and mechanical extremes for advanced applications (e.g. fission & fusion reactors, space applications, etc.), design, prediction and control of advanced materials beyond current material designs become paramount. Here, through a combined experimental and simulation methodology, we design a nanocrystalline refractory high entropy alloy (RHEA) system. Compositions assessed under extreme environments and in situ electron-microscopy reveal both high thermal stability and radiation resistance. We observe grain refinement under heavy ion irradiation and resistance to dual-beam irradiation and helium implantation in the form of low defect generation and evolution, as well as no detectable grain growth. The experimental and modeling results-showing a good agreement-can be applied to design and rapidly assess other alloys subjected to extreme environmental conditions.

Heat-stable spores of carotenoid-producing Bacillus marisflavi and non-pigmented Bacillus subtilis cooperatively promote growth, quality, and gut microbiota of white-leg shrimp.

We evaluated the benefits of heat-stable carotenoid-producing Bacillus marisflavi SH8 spores individually and in combination with non-pigmented Bacillus subtilis SH23 spores on growth, colour change, nutritional content, innate immunity, and gut microbiota of white-leg shrimp. White-leg shrimp (Litopenaeus vannamei; n = 30 per tank; 2 tanks per group) were provided feed without (control group) or with SH8, SH23, or mixed spores (total, 1 × 106 cfu/g pellet) for 28 d. The SH8 and SH8-23 combination groups had significantly higher specific growth rates (9.6 and 11.0%), improved red-colour score (4 scores), astaxanthin concentration (1.8- and 2.3-fold), lipid contents (30 and 50%), and superoxidase dismutase activity (8.5 and 12.3%) than that of the control group. Analysis of shrimp's gut microbiome using 16S rRNA metagenome sequencing revealed increased abundance of four useful species and reduced abundance of four harmful species in the combination group than in the control group. Heat-stable Bacillus spore combination improved growth parameters, nutrient content, red-colour score, live counts, and abundance of useful bacteria in the gut of L. vannamei. This is the first study to show the benefits of combining highly heat-stable pigmented and non-pigmented Bacillus spores and their possible mechanisms in a shrimp model.

Measuring shared decision-making in younger and older adults with depression.

BACKGROUND: This study examined the performance of the shared decision-making (SDM) Process scale in patients with depression, compared alternative wording of two items in the scale and explored performance in younger adults. METHODS: A web-based non-probability panel of respondents with depression aged 18-39 (younger) or 40-75 (older) who talked with a health-care provider about starting or stopping treatment for depression in the past year were surveyed. Respondents completed one of two versions of the SDM Process scale that differed in the wording of pros and cons items and completed measures of decisional conflict, decision regret and who made the decision (mainly the respondent, mainly the provider or together). A subset of respondents completed a retest survey by 1 week. We examined how version and age group impacted SDM Process scores and calculated construct validity and retest reliability. We hypothesized that patients with higher SDM Process scores would show less decisional conflict using the SURE scale (range = 0-4); top score = no conflict versus other and less regret (range 1-4; higher scores indicated more regret). RESULTS: The sample (N = 494) was majority White, non-Hispanic (82%) and female (72%), 48% were younger and 23% had a high school education or less. SDM Process scores did not differ by version (P = 0.09). SDM Process scores were higher for younger respondents (M = 2.6, SD = 1.0) than older respondents (M = 2.3, SD = 1.1; P = 0.001). Higher SDM Process scores were also associated with no decisional conflict (M = 2.6, SD = 0.99 vs. M = 2.1, SD = 1.2; P < 0.001) and less decision regret (r = -0.18, P < 0.001). Retest reliability was intraclass correlation coefficient = 0.81. CONCLUSIONS: The SDM Process scale demonstrated validity and retest reliability in younger adults, and changes to item wording did not impact scores. Although younger respondents reported more SDM, there is room for improvement in SDM for depression treatment decisions.

3D reconstruction of bird flight trajectories using a single video camera.

Video cameras are finding increasing use in the study and analysis of bird flight over short ranges. However, reconstruction of flight trajectories in three dimensions typically requires the use of multiple cameras and elaborate calibration procedures. We present an alternative approach that uses a single video camera and a simple calibration procedure for the reconstruction of such trajectories. The technique combines prior knowledge of the wingspan of the bird with a camera calibration procedure that needs to be used only once in the lifetime of the system. The system delivers the exact 3D coordinates of the position of the bird at the time of every full wing extension and uses interpolated height estimates to compute the 3D positions of the bird in the video frames between successive wing extensions. The system is inexpensive, compact and portable, and can be easily deployed in the laboratory as well as the field.

Updated evidence of the safety and efficacy of the miniaturized percutaneous nephrolithotomy with holmium laser lithotripsy for the treatment of recurrent nephrolithiasis.

OBJECTIVE: The objective of the present communication is to report the safety and efficacy of applying miniaturized percutaneous nephrolithotomy (mini-PCNL) in Vietnamese patients with recurrent kidney stones using a miniature nephroscope through a 18F metal access sheath. PATIENTS AND METHODS: We performed a retrospective cohort study of patients who were diagnosed with recurrent nephrolithiasis and underwent mini-PCNL after previous treatments between 2017 and 2020. Clinical profile, preoperative characteristics, intraoperative events, and postoperative outcomes were obtained from the hospital database. Descriptive statistics were used for the whole analysis. RESULTS: Of 89 eligible patients, 54 were male. The mean age was 53.4 years. Mean stone size was 24.9 mm. 37 patients had right side stone, 39 had left side stone, and 13 had stone on both sides. Pain in the flanks and back was the most symptom in our patients (n = 87, 97.8%), followed by hematuria (n = 13, 14.6%), dysuria (n = 8, 8.9%), fever (n = 7, 7.9%), acute renal colic (n = 5, 5.6%), and pyuria in 2 patients. 27 had complex stones, and the remaining stone position included renal pelvis (21, 23.6%), upper calyx (15, 16.9%), lower calyx (14, 15.7%), and middle calyx (12, 13.5%). 35 of them had grade I, 27 grade II, 8 grade III and 2 grade IV of hydronephrosis. Renal failure was documented in 12 patients (13.5%). 18 patients were performed with 2-times punctures (20.2%) and 13 with 3-times punctures (14.6%). We used one percutaneous tract in 79 patients and the remaining 10 were performed with two tracts. The upper, middle, and lower calyx was the site of puncture access in 6 (6.7%), 73 (82.0%), and 10 (11.3%) cases, respectively. 3 patients had bleeding requiring intraoperative blood transfusion and 2 were converted to open surgery. Intraoperative parameters recorded percutaneous puncture duration of 20.9 minutes (5-50), and total operative duration of 112.9 minutes (40-240). 7 patients developed secondary bleeding after surgery, besides 5 cases of fever, 2 cases of septic shock and one case of drainage failure. Early stone-free rate was 89.9% and this rate was 94.4% for patients after one month of mini-PCNL. Mean duration of ureteral catheter circulation was 2.7 days (2-20), mean length of postoperative hospitalization was 6.6 days (4-25) and mean total hospital stay was 12.9 days (7-28). CONCLUSIONS: Present results show the safety of mini-PCNL with respect to recurrent nephrolithiasis. Our updated evidence may provide appropriate modified approaches that aim at reducing the risk of recurrent kidney stone development.

Posterior surgical approach for the treatment of lower cervical spine injury with spinal cord paralysis: high postoperative mortality in resource-scare setting.

OBJECTIVE: This report aimed to characterize clinical and imaging characteristics and outcomes of the patients with lower cervical spine injury combined with spinal cord paralysis who underwent posterior cervical spine surgery. PATIENTS AND METHODS: Between January 2019 and December 2020, a retrospective evaluation of prospectively collected data at one institution was conducted. We included all patients who were diagnosed with subaxial cervical spine injuries (C3-7), had spinal cord paralysis, and underwent posterior cervical spine surgery. Clinical profile, preoperative characteristics, intraoperative data, and postoperative outcomes were retrieved from prospective patients' medical records and computerized database. RESULTS: Among 70 selected patients, most were male (66, 94.29%) and the average age was 48.41 ± 14.33 years. Most of them worked in agriculture (90.4%). Clinical symptoms included neck pain (58, 82.86%), cervical radiculopathy (50, 71.43%), loss of sensation (44, 62.86%), and decreased sensation (21, 30.00%). The most frequent cervical spinal injuries involved C5 (28.57%), followed by C7 (14.29%). Circular muscle dysfunction was present in 65 (92.86%) patients. Early complications included respiratory failure (12.85%), pneumonia (11.42%), bedsores (8.57%), and urinary tract infection (7.14%). Common late complications included movement disorder (48.21%), muscle weakness and stiffness (37.50%), sensory disturbances (32.14%), urinary tract infection (17.86%), bedsores (16.07%), and pneumonia (5.36%). Patients after surgery and at follow-up had a significant improvement compared to preoperative assessment according to the AIS classification, and recovery of smooth muscle. Three patients died within 1 month following surgery, 3 within 1-3 month(s), 2 within 3-6 months, and 1 case beyond 6 months. CONCLUSIONS: In hospital-based clinical condition with limited practice approach, our study indicated specific clinical and imaging characteristics of Vietnamese patients with lower cervical spine injury combined with spinal cord paralysis. With high postoperative mortality rate, commonly late complications after posterior cervical spine surgical approach were pain and difficulty in neck movement, muscle weakness and stiffness, and nerve root pain.

Diagnostic value of cerebral vasospasm by transcranial doppler ultrasound in Vietnamese patients with subarachnoid hemorrhage.

OBJECTIVE: Although the application of transcranial Doppler (TCD) ultrasonography in clinical diagnosis of cerebral vasospasm is popular in clinical practice in Vietnam, available evidence of the predictive value of vasospasm on TCD in the literature was mostly reported from large institutions in developed countries. Hence, this study was conducted to evaluate the value of TCD ultrasonography in the diagnosis of vasospasm in patients with subarachnoid hemorrhage (SAH) in Vietnam. PATIENTS AND METHODS: This is a prospective observational study of all aneurysmal SAH patients consecutively admitted to a single center between 2008 and December 2011. TCD and 64-slice computed tomographic angiography (CTA) were used to cerebral vasospasm in SAH patients. RESULTS: 316 patients were analyzed (mean age = 52.97±12.27 years, 52.2% males). There were statistically significant difference rates of the cerebral vasospasm by Hunt and Hess Classification and Fisher classification (p <0.01). The proportion of the patients with cerebral vasospasm who were diagnosed exactly by TCD was 95.2%, while the proportion of the patients without cerebral vasospasm diagnosed exactly was 91.5%. TCD predictive diagnostic value was the highest, with the sensitivity of 0.95 (95% CI: 0.91-0.98), specificity of 0.91 (95% CI: 0.85-0.96), positive predictive value of 0.94 (5% CI: 0.90-0.97) and negative predictive value of 0.93 (95 CI: 0.87-0.97). Hemiplegia was the clinical symptom with the highest diagnostic value with the sensitivity of 0.34 (95% CI: 0.27-0.41), specificity of 0.92 (95% CI: 0.86-0.96), positive predictive value of 0.86 (95% CI: 0.76-0.93) and negative predictive value of 0.49 (95% CI: 0.41-0.54). CONCLUSIONS: Evidence of vasospasm diagnosis on TCD ultrasonography was found with high accuracy. Current study enables to suggest the wide application of TCD in Vietnam health facilities from central to grassroots levels instead of the CTA use.

Prevalence of depression among heart failure inpatients and its associated socio-demographic factors: implications for personal-and family-based treatment management in health facilities in Vietnam.

OBJECTIVE: Our objective is to identify the prevalence of depression among inpatients with heart failure (HF), and to ascertain the factors associated with the depression from a wide spectrum of sociodemographic variables. MATERIALS AND METHODS: We conducted a hospital-based cross-sectional survey of prospectively collected data in inpatients with a diagnosis of HF at Vietnam National Heart Institute, Bach Mai Hospital (Hanoi, Vietnam) from July 2020 to July 2021. A sample size of 128 inpatients with HF was finally included. Primary outcome variable was depression ICD-10. RESULTS: The mean age was 62.34 (SD = 14.76). The sex ratio was 66 males to 62 females. The overall prevalence of depression ICD-10 was 46.88% among HF inpatients. The proportion of the depressed patients fluctuated between 37.21% and 83.33% by NYHA heart failure classification. Compared to the depressive prevalence among patients with NYHA class II, the odds were 8.43 times higher for those with NYHA class IV (OR univariate 8.43; 95% CI 1.63-43.46). Patient's age was significantly associated with increased prevalence of depression (OR multivariate 1.07; 95% CI 1.003-1.14). It was significantly higher odds of depression in HF patients who felt sadness after a diagnosis of an illness (OR multivariate 18.02, 95% CI: 4.21-77.08). Individuals with higher household economic status were less likely to be diagnosed with depression compared to those with lower household economic status (OR multivariate 0.15, 95% CI: 0.02-0.92). The odds of depression were significantly higher in HF patients who reported family conflict (OR multivariate 23.45, 95% CI: 1.29-423.55), and in those having the loss of a close family member (OR multivariate 38.62, 95% CI: 1.41-1055.98). CONCLUSIONS:   The prevalence of depression by ICD-10 was relatively high. Age of patient, sadness after a diagnosis of an illness, household economic status, family conflict and loss of a close family member were significantly associated with the depression among HF inpatients. Present results suggest a need for a disease management program addressing both psychological and HF aspects with the aim of improving health outcomes for the inpatients in Vietnam health facilities.

A novel modification of frozen elephant trunk technique: unique protocol from one institution.

OBJECTIVE: We aim to present a novel surgical technique of Frozen Elephant Trunk (FET) to treat complex thoracic aortic diseases in one stage and report its short-term outcomes. PATIENTS AND METHODS: Between December 2019 and 30 April 2021, twenty-five patients underwent FET operation at Viet Duc University Hospital. The mean age of the patients was 55.9 (±9.9, range 33-72) years. Eighteen (72%) of the patients were men. Thoracic aortic aneurysm was presented in three (12%) patients. Among seventeen (68%) of the patients undergoing the aortic dissection, eleven (44%) were treated acute type A aortic dissection. Type A intramural hematoma was presented in three (12%) patients. Four (16%) of the patients had undergone previous aortic operations, four (16%) of them had Marphan syndrome and two (11.1%) of them had stage 3 chronic kidney disease. All patients underwent FET procedure by unique protocol. Brain protection was achieved by antegrade bilateral selective cerebral perfusion and moderate hypothermia (28°C) in all cases; besides cerebral tissue oximetry monitoring was used to control brain oxygenation. RESULTS: There were no perioperative deaths, and all patients are still alive during mild-term follow-up period. Sixteen (88.9%) patients received isolated FET, while a Bentall procedure during FET was performed in two (8%) patients and right coronary artery bypass was in one (4%) case. The duration of cardiopulmonary bypass, cross-clamping, circulatory arrest, and total operation were 176.7 (±48.1, range 102-330), 106 (±39.8, range 63-205), 32.7 (±9.6, range 20-58), and 365.6 (±53.6, range 270-480) min, respectively. There was no bleeding following surgery. Prolonged ventilation required tracheotomy was documented in two (8%) patients, hemodialysis caused acute renal failure was in five (20%) patients, cerebral shock was in one (4%) patient, and type 1A endoleak in 2 (8%) patients. CONCLUSIONS: Our modification of FET technique was feasible, effective, and safe, with good postoperative outcomes.

Appearance of reassortant European avian-origin H1 influenza A viruses of swine in Vietnam.

Three subtypes-H1N1, H1N2 and H3N2-of influenza A viruses of swine (IAVs-S) are currently endemic in swine worldwide, but there is considerable genotypic diversity among each subtype and limited geographical distribution. Through IAVs-S monitoring in Vietnam, two H1N2 influenza A viruses were isolated from healthy pigs in Ba Ria-Vung Tau Province, Southern Vietnam, on 2 December 2016. BLAST and phylogenetic analyses revealed that their HA and NA genes were derived from those of European avian-like H1N2 IAVs-S that contained avian-origin H1 and human-like N2 genes, and were particularly closely related to those of IAVs-S circulating in the Netherlands, Germany or Denmark. In addition, the internal genes of these Vietnamese isolates were derived from human A(H1N1)pdm09 viruses, suggesting that the Vietnamese H1N2 IAVs-S are reassortants between European H1N2 IAVs-S and human A(H1N1)pdm09v. The appearance of European avian-like H1N2 IAVs-S in Vietnam marks their first transmission outside Europe. Our results and statistical analyses of the number of live pigs imported into Vietnam suggest that the European avian-like H1N2 IAVs-S may have been introduced into Vietnam with their hosts through international trade. These findings highlight the importance of quarantining imported pigs to impede the introduction of new IAVs-S.

Solving the chemical master equation by a fast adaptive finite state projection based on the stochastic simulation algorithm.

The mathematical framework of the chemical master equation (CME) uses a Markov chain to model the biochemical reactions that are taking place within a biological cell. Computing the transient probability distribution of this Markov chain allows us to track the composition of molecules inside the cell over time, with important practical applications in a number of areas such as molecular biology or medicine. However the CME is typically difficult to solve, since the state space involved can be very large or even countably infinite. We present a novel way of using the stochastic simulation algorithm (SSA) to reduce the size of the finite state projection (FSP) method. Numerical experiments that demonstrate the effectiveness of the reduction are included.

Physiological implications of genomic state in parthenogenetic lizards of reciprocal hybrid origin.

Parthenogenesis often evolves in association with hybridization, but the associated ecological consequences are poorly understood. The Australian gecko Heteronotia binoei is unusual because triploid parthenogenesis evolved through reciprocal crosses between two sexual lineages, resulting in four possible cytonuclear genotypes. In this species complex, we compared the performance of these parthenogenetic genotypes with their sexual progenitors for a suite of physiological traits (metabolic rate, thermal tolerance, locomotor performance, and in vitro activity and gene sequence divergence of a cytonuclear metabolic pathway, cytochrome C oxidase). Mass-specific metabolic rate scaled differently with body mass for parthenogens and sexuals, while heat tolerance provided the only evidence for cytonuclear incompatibility in hybrid parthenogens. The most prominent phenotypic effects were attributable to nuclear genome dosage. Overall, our results suggest that the hybrid/polyploidy origin of parthenogenetic H. binoei has had surprisingly few negative fitness consequences and may have produced a broader overall niche for the species.

Notch3 cooperates with the EGFR pathway to modulate apoptosis through the induction of bim.

Notch signaling is a highly conserved pathway important for normal embryonic development and cancer. We previously demonstrated a role for Notch3 in lung cancer pathogenesis. Notch3 inhibition resulted in tumor apoptosis and growth suppression. In vitro, these effects were enhanced when the epidermal growth factor receptor (EGFR) pathway was also inhibited, suggesting significant cross-talk between the two pathways. How Notch3 and epidermal growth factor receptor-mitogen-activated protein kinase (EGFR-MAPK) pathways cooperate in modulating apoptosis is not yet known. In this study, we provide evidence that Notch3 regulates Bim, a BH-3-only protein, via MAPK signaling. Furthermore, loss of Bim expression prevents tumor apoptosis induced by Notch3 inhibition. Using gamma-secretase inhibitor and erlotinib in a xenograft model, Bim induction and tumor inhibition were observed to be enhanced compared with either agent alone, consistent with our previous observation of significant synergism between Notch and EGFR-ras-MAPK signaling. Thus, our data support the hypothesis that Notch3 not only has a crucial role in lung cancer through regulating apoptosis, but also cooperates with the EGFR-MAPK pathway in modulating Bim.

Structured antiretroviral treatment interruptions in chronically HIV-1-infected subjects.

The risks and benefits of structured treatment interruption (STI) in HIV-1-infected subjects are not fully understood. A pilot study was performed to compare STI with continuous highly active antiretroviral therapy (HAART) in chronic HIV-1-infected subjects with HIV-1 plasma RNA levels (VL) <400 copies per ml and CD4(+) T cells >400 per microl. CD4(+) T cells, VL, HIV-1-specific neutralizing antibodies, and IFN-gamma-producing HIV-1-specific CD8(+) and CD4(+) T cells were measured in all subjects. STIs of 1-month duration separated by 1 month of HAART, before a final 3-month STI, resulted in augmented CD8(+) T cell responses in all eight STI subjects (P = 0.003), maintained while on HAART up to 22 weeks after STI, and augmented neutralization titers to autologous HIV-1 isolate in one of eight subjects. However, significant decline of CD4(+) T cell count from pre-STI level, and VL rebound to pre-HAART baseline, occurred during STI (P = 0.001 and 0.34, respectively). CD4(+) T cell counts were regained on return to HAART. Control subjects (n = 4) maintained VL <400 copies per ml and stable CD4(+) T cell counts, and showed no enhancement of antiviral CD8(+) T cell responses. Despite increases in antiviral immunity, no control of VL was observed. Future studies of STI should proceed with caution.

Neutralizing antibodies associated with viremia control in a subset of individuals after treatment of acute human immunodeficiency virus type 1 infection.

Immediate treatment of acute human immunodeficiency virus type 1 (HIV-1) infection has been associated with subsequent control of viremia in a subset of patients after therapy cessation, but the immune responses contributing to control have not been fully defined. Here we examined neutralizing antibodies as a correlate of viremia control following treatment interruption in HIV-1-infected individuals in whom highly active antiretriviral therapy (HAART) was initiated during early seroconversion and who remained on therapy for 1 to 3 years. Immediately following treatment interruption, neutralizing antibodies were undetectable with T-cell-line adapted strains and the autologous primary HIV-1 isolate in seven of nine subjects. Env- and Gag-specific antibodies as measured by enzyme-linked immunosorbent assay were also low or undetectable at this time. Despite this apparent poor maturation of the virus-specific B-cell response during HAART, autologous neutralizing antibodies emerged rapidly and correlated with a spontaneous downregulation in rebound viremia following treatment interruption in three subjects. Control of rebound viremia was seen in other subjects in the absence of detectable neutralizing antibodies. The results indicate that virus-specific B-cell priming occurs despite the early institution of HAART, allowing rapid secondary neutralizing-antibody production following treatment interruption in a subset of individuals. Since early HAART limits viral diversification, we hypothesize that potent neutralizing-antibody responses to autologous virus are able to mature and that in some persons these responses contribute to the control of plasma viremia after treatment cessation.

Induction of neutralizing antibodies and gag-specific cellular immune responses to an R5 primary isolate of human immunodeficiency virus type 1 in rhesus macaques.

The ability to generate antibodies that cross-neutralize diverse primary isolates is an important goal for human immunodeficiency virus type 1 (HIV-1) vaccine development. Most of the candidate HIV-1 vaccines tested in humans and nonhuman primates have failed in this regard. Past efforts have focused almost entirely on the envelope glycoproteins of a small number of T-cell line-adapted strains of the virus as immunogens. Here we assessed the immunogenicity of noninfectious virus-like particles (VLP) consisting of Gag, Pro (protease), and Env from R5 primary isolate HIV-1(Bx08). Immunogens were delivered to rhesus macaques in the form of either purified VLP, recombinant DNA and canarypox (ALVAC) vectors engineered to express VLP, or a combination of these products. Seroconversion to Gag and Pro was detected in all of the immunized animals. Antibodies that could neutralize HIV-1(Bx08) were detected in animals that received (i) coinoculations with DNA(Bx08) and VLP(Bx08), (ii) DNA(Bx08) followed by ALVAC(Bx08) boosting, and (iii) VLP(Bx08) alone. The neutralizing antibodies were highly strain specific despite the fact that they did not appear to be directed to linear epitopes in the V3 loop. Virus-specific cellular immune responses also were generated, as judged by the presence of Gag-specific gamma interferon (IFN-gamma)-producing cells. These cellular immune responses required the inclusion of DNA(Bx08) in the immunization modality, since few or no IFN-gamma-producing cells were detected in animals that received either VLP(Bx08) or ALVAC(Bx08) alone. The results demonstrate the feasibility of generating neutralizing antibodies and cellular immune responses that target an R5 primary HIV-1 isolate by vaccination in primates.

Competence development by Haemophilus influenzae is regulated by the availability of nucleic acid precursors.

DNA uptake by naturally competent bacteria provides cells with both genetic information and nucleotides. In Haemophilus influenzae, competence development requires both cAMP and an unidentified signal arising under starvation conditions. To investigate this signal, competence induction was examined in media supplemented with nucleic acid precursors. The addition of physiological levels of AMP and GMP reduced competence 200-fold and prevented the normal competence-induced transcription of the essential competence genes comA and rec-2. The rich medium normally used for growth allows only limited competence. Capillary electrophoresis revealed only a subinhibitory amount of AMP and no detectable GMP, and the addition of AMP or GMP to this medium also reduced competence 20- to 100-fold. Neither a functional stringent response system nor a functional phosphoenolpyruvate:glycose phosphotransferase system (PTS) was found to be required for purine-mediated repression. Added cAMP partially restored both transcription of competence genes and competence development, suggesting that purines may reduce the response to cAMP. Potential binding sites for the PurR repressor were identified in several competence genes, suggesting that competence is part of the PUR regulon. These observations are consistent with models of competence regulation, in which depleted purine pools signal the need for nucleotides, and support the hypothesis that competence evolved primarily for nucleotide acquisition.

Analysis of gamma-carboxyglutamic acid content of protein, urine, and plasma by capillary electrophoresis and laser-induced fluorescence.

When the properties of an analyte are known, the separation system can be designed to make the analyte of interest migrate at either a much faster or a much slower velocity compared to other molecules in the sample matrix. A simple and sensitive method to analyze the gamma-carboxyglutamic acid (Gla) content of protein, urine, and plasma was developed using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). The separation method is designed according to the specific properties of three amino acids of interest. The number of Gla residues from three vitamin K-dependent proteins were estimated by quantifying the amount of fluorescein thiocarbamyl derivative of Gla after alkaline hydrolysis and fluorescein isothiocyanate labeling. Human prothrombin, blood coagulation factor X, and bovine osteocalcin were calculated to have 10.0 +/- 0.7, 11.0 +/- 0.6, and 2.1 +/- 0.1 Gla residues per mole of protein, respectively, which agreed well with amino acid sequencing data. The analysis of free Gla content in urine and plasma was also demonstrated by this method. It was demonstrated that submicrograms of protein can be characterized by CE-LIF.