DNA-Diffusion: Leveraging Generative Models for Controlling Chromatin Accessibility and Gene Expression via Synthetic Regulatory Elements.

The challenge of systematically modifying and optimizing regulatory elements for precise gene expression control is central to modern genomics and synthetic biology. Advancements in generative AI have paved the way for designing synthetic sequences with the aim of safely and accurately modulating gene expression. We leverage diffusion models to design context-specific DNA regulatory sequences, which hold significant potential toward enabling novel therapeutic applications requiring precise modulation of gene expression. Our framework uses a cell type-specific diffusion model to generate synthetic 200 bp regulatory elements based on chromatin accessibility across different cell types. We evaluate the generated sequences based on key metrics to ensure they retain properties of endogenous sequences: transcription factor binding site composition, potential for cell type-specific chromatin accessibility, and capacity for sequences generated by DNA diffusion to activate gene expression in different cell contexts using state-of-the-art prediction models. Our results demonstrate the ability to robustly generate DNA sequences with cell type-specific regulatory potential. DNA-Diffusion paves the way for revolutionizing a regulatory modulation approach to mammalian synthetic biology and precision gene therapy.

Computer aided drug design in the development of proteolysis targeting chimeras.

Proteolysis targeting chimeras represent a class of drug molecules with a number of attractive properties, most notably a potential to work for targets that, so far, have been in-accessible for conventional small molecule inhibitors. Due to their different mechanism of action, and physico-chemical properties, many of the methods that have been designed and applied for computer aided design of traditional small molecule drugs are not applicable for proteolysis targeting chimeras. Here we review recent developments in this field focusing on three aspects: de-novo linker-design, estimation of absorption for beyond-rule-of-5 compounds, and the generation and ranking of ternary complex structures. In spite of this field still being young, we find that a good number of models and algorithms are available, with the potential to assist the design of such compounds in-silico, and accelerate applied pharmaceutical research.

Temporal Dynamics of Subclinical Malaria in Different Transmission Zones of Myanmar.

Countries in the Greater Mekong Subregion have committed to eliminate Plasmodium falciparum malaria by 2025. Subclinical malaria infections that can be detected by highly sensitive polymerase chain reaction (PCR) testing in asymptomatic individuals represent a potential impediment to this goal, although the extent to which these low-density infections contribute to transmission is unclear. To understand the temporal dynamics of subclinical malaria in this setting, a cohort of 2,705 participants from three epidemiologically distinct regions of Myanmar was screened for subclinical P. falciparum and P. vivax infection using ultrasensitive PCR (usPCR). Standard rapid diagnostic tests (RDTs) for P. falciparum were also performed. Individuals who tested positive for malaria by usPCR were followed for up to 12 weeks. Regression analysis was performed to estimate whether the baseline prevalence of infection and the count of repeated positive tests were associated with demographic, behavioral, and clinical factors. At enrollment, the prevalence of subclinical malaria infection measured by usPCR was 7.7% (1.5% P. falciparum monoinfection, 0.3% mixed P. falciparum and P. vivax, and 6.0% P. vivax monoinfection), while P. falciparum prevalence measured by RDT was just 0.2%. Prevalence varied by geography and was higher among older people and in those with outdoor exposure and travel. No difference was observed in either the prevalence or count of subclinical infection by time of year, indicating that even in low-endemicity areas, a reservoir of subclinical infection persists year-round. If low-density infections are shown to represent a significant source of transmission, identification of high-risk groups and locations may aid elimination efforts.

Molecular epidemiology of resistance to antimalarial drugs in the Greater Mekong subregion: an observational study.

BACKGROUND: The Greater Mekong subregion is a recurrent source of antimalarial drug resistance in Plasmodium falciparum malaria. This study aimed to characterise the extent and spread of resistance across this entire region between 2007 and 2018. METHODS: P falciparum isolates from Myanmar, Thailand, Laos, and Cambodia were obtained from clinical trials and epidemiological studies done between Jan 1, 2007, and Dec 31, 2018, and were genotyped for molecular markers (pfkelch, pfcrt, pfplasmepsin2, and pfmdr1) of antimalarial drug resistance. Genetic relatedness was assessed using microsatellite and single nucleotide polymorphism typing of flanking sequences around target genes. FINDINGS: 10 632 isolates were genotyped. A single long pfkelch Cys580Tyr haplotype (from -50 kb to +31·5 kb) conferring artemisinin resistance (PfPailin) now dominates across the eastern Greater Mekong subregion. Piperaquine resistance associated with pfplasmepsin2 gene amplification and mutations in pfcrt downstream of the Lys76Thr chloroquine resistance locus has also developed. On the Thailand-Myanmar border a different pfkelch Cys580Tyr lineage rose to high frequencies before it was eliminated. Elsewhere in Myanmar the Cys580Tyr allele remains widespread at low allele frequencies. Meanwhile a single artemisinin-resistant pfkelch Phe446Ile haplotype has spread across Myanmar. Despite intense use of dihydroartemisinin-piperaquine in Kayin state, eastern Myanmar, both in treatment and mass drug administrations, no selection of piperaquine resistance markers was observed. pfmdr1 amplification, a marker of resistance to mefloquine, remains at low prevalence across the entire region. INTERPRETATION: Artemisinin resistance in P falciparum is now prevalent across the Greater Mekong subregion. In the eastern Greater Mekong subregion a multidrug resistant P falciparum lineage (PfPailin) dominates. In Myanmar a long pfkelch Phe446Ile haplotype has spread widely but, by contrast with the eastern Greater Mekong subregion, there is no indication of artemisinin combination therapy (ACT) partner drug resistance from genotyping known markers, and no evidence of spread of ACT resistant P falciparum from the east to the west. There is still a window of opportunity to prevent global spread of ACT resistance. FUNDING: Thailand Science Research and Innovation, Initiative 5%, Expertise France, Wellcome Trust.

Uterine artery pseudoaneurysm: A case of late intra-abdominal haemorrhage after caesarean section.

Uterine artery pseudoaneurysm (UAP) is a rare acquired vascular malformation associated with vaginal bleeding or intraabdominal haemorrhage occurring after pelvic surgery. Pseudoaneurysm may present with delayed, severe haemorrhage after a seemingly uncomplicated initial postoperative period. Treatment is therefore necessary to prevent further complications. We describe here a case of a 32-year-old mother, who presented with abdominal pain and intraabdominal bleeding, 20 days after Caesarean Section. Computerised Tomography (CT) scan showed the presence of haemoperitoneum, suggestive of pseudoaneurysm at the right cervical artery which was successfully managed with emergency angiographic embolisation.

Using Cellulolytic Nitrogen Fixing Bacterium, Azomonas agilis for Effective Degradation of Agricultural Residues.

INTRODUCTION: Azomonas agilis, a nitrogen-fixing bacterium, was isolated from rhizospheric soil in central Myanmar. METHODS & MATERIALS: The nitrogen-fixing activity of this bacterium was detected by plate screening method using glucose nitrogen free mineral medium and ammonium test-kit Cellulolytic activity was screened by plat assay and detected by Dinitrosalicyclic acid method (DNS). RESULTS & DISCUSSION: The isolated A. agilis grew in media containing 3-12% of NaCl, although the growth became poor when NaCl concentrations increased. Among various carbon sources, sucrose was the best source for ammonium accumulation of this bacterium, whereas arabinose was not the suitable carbon source. Although the nitrogen-fixing activity of A. agilis was highest after one week incubation, cellulase enzyme production was highest after 2-3 days of incubation. It was observed that cellulase enzyme activity of A. agilis for cellulose and sodium carboxymethyl cellulose (CMC) was almost the same. Three agricultural wastes were used to detect the cellulase enzyme activity of A. agilis, cellulase activity was better on filter paper as a substrate when compared to rice-straw and sawdust. CONCLUSION: So, the isolated A. agilis has high potential as an effective bacterial strain to use in sustainable agriculture and degradation of some agricultural residues.

Preventing emergence of drug resistant tuberculosis in Myanmar's transitioning health system.

Multidrug-resistant tuberculosis (MDR-TB) is a particular threat to the populations of resource-limited countries. Although inadequate treatment of TB has been identified as a major underlying cause of drug resistance, essential information to inform changes in health service delivery and policy is missing. We investigate factors that may be driving the emergence of MDR-TB in Myanmar, a country where investment and health system reforms are ongoing to address the unexplained, high occurrence of MDR-TB. We conducted a multi-centre, retrospective case-control study in 10 townships across Yangon. Cases were 202 GeneXpert-confirmed MDR-TB patients with a history of prior first-line treatment for TB. Controls were 404 previously untreated smear-microscopy confirmed TB patients who had no evidence of resistance to anti-TB drugs. Information on patient and health service factors was collected through face-to-face patient interviews and hospital record reviews. Multivariable logistic regression analysis indicated that the following TB patient groups are at higher risk of developing MDR-TB after initial TB treatment: those who have diabetes (aOR 2.10; 95% CI 1.17-3.76), those who missed taking drugs during the initial treatment more than once weekly (aOR 2.35; 95% CI 1.18-4.65) and those with a higher socioeconomic (aOR 1.99; 95% CI 1.09-3.63) or educational status (aOR 1.78; 95% CI1.01-3.13). Coinciding with a surge in funding to improve health in Myanmar, this study identifies practices of patients and healthcare organizations that can be addressed, and high-risk TB patient groups that can be prioritized for treatment support. Specifically, the study shows that TB patients who experience frequent, short interruptions in treatment and those with diabetes may require enhanced treatment support and monitoring by health services in order to prevent further generation of drug resistance.

Amniocentesis and chorionic villus sampling in HIV-infected pregnant women: a multicentre case series.

OBJECTIVES: To assess in pregnant women with HIV the rates of amniocentesis and chorionic villus sampling (CVS), and the outcomes associated with such procedures. DESIGN: Observational study. Data from the Italian National Program on Surveillance on Antiretroviral Treatment in Pregnancy were used. SETTING: University and hospital clinics. POPULATION: Pregnant women with HIV. METHODS: Temporal trends were analysed by analysis of variance and by the Chi-square test for trend. Quantitative variables were compared by Student's t-test and categorical data by the Chi-square test, with odds ratios and 95% confidence intervals calculated. MAIN OUTCOME MEASURES: Rate of invasive testing, intrauterine death, HIV transmission. RESULTS: Between 2001 and 2015, among 2065 pregnancies in women with HIV, 113 (5.5%) had invasive tests performed. The procedures were conducted under antiretroviral treatment in 99 cases (87.6%), with a significant increase over time in the proportion of tests performed under highly active antiretroviral therapy (HAART) (100% in 2011-2015). Three intrauterine deaths were observed (2.6%), and 14 pregnancies were terminated because of fetal anomalies. Among 96 live newborns, eight had no information available on HIV status. Among the remaining 88 cases with either amniocentesis (n = 75), CVS (n = 12), or both (n = 1), two HIV transmissions occurred (2.3%). No HIV transmission occurred among the women who were on HAART at the time of invasive testing, and none after 2005. CONCLUSIONS: The findings reinforce the assumption that invasive prenatal testing does not increase the risk of HIV vertical transmission among pregnant women under suppressive antiretroviral treatment. TWEETABLE ABSTRACT: No HIV transmission occurred among women who underwent amniocentesis or CVS under effective anti-HIV regimens.

Optimal health and disease management using spatial uncertainty: a geographic characterization of emergent artemisinin-resistant Plasmodium falciparum distributions in Southeast Asia.

BACKGROUND: Artemisinin-resistant Plasmodium falciparum malaria parasites are now present across much of mainland Southeast Asia, where ongoing surveys are measuring and mapping their spatial distribution. These efforts require substantial resources. Here we propose a generic 'smart surveillance' methodology to identify optimal candidate sites for future sampling and thus map the distribution of artemisinin resistance most efficiently. METHODS: The approach uses the 'uncertainty' map generated iteratively by a geostatistical model to determine optimal locations for subsequent sampling. RESULTS: The methodology is illustrated using recent data on the prevalence of the K13-propeller polymorphism (a genetic marker of artemisinin resistance) in the Greater Mekong Subregion. CONCLUSION: This methodology, which has broader application to geostatistical mapping in general, could improve the quality and efficiency of drug resistance mapping and thereby guide practical operations to eliminate malaria in affected areas.

Spread of artemisinin-resistant Plasmodium falciparum in Myanmar: a cross-sectional survey of the K13 molecular marker.

BACKGROUND: Emergence of artemisinin resistance in southeast Asia poses a serious threat to the global control of Plasmodium falciparum malaria. Discovery of the K13 marker has transformed approaches to the monitoring of artemisinin resistance, allowing introduction of molecular surveillance in remote areas through analysis of DNA. We aimed to assess the spread of artemisinin-resistant P falciparum in Myanmar by determining the relative prevalence of P falciparum parasites carrying K13-propeller mutations. METHODS: We did this cross-sectional survey at malaria treatment centres at 55 sites in ten administrative regions in Myanmar, and in relevant border regions in Thailand and Bangladesh, between January, 2013, and September, 2014. K13 sequences from P falciparum infections were obtained mainly by passive case detection. We entered data into two geostatistical models to produce predictive maps of the estimated prevalence of mutations of the K13 propeller region across Myanmar. FINDINGS: Overall, 371 (39%) of 940 samples carried a K13-propeller mutation. We recorded 26 different mutations, including nine mutations not described previously in southeast Asia. In seven (70%) of the ten administrative regions of Myanmar, the combined K13-mutation prevalence was more than 20%. Geospatial mapping showed that the overall prevalence of K13 mutations exceeded 10% in much of the east and north of the country. In Homalin, Sagaing Region, 25 km from the Indian border, 21 (47%) of 45 parasite samples carried K13-propeller mutations. INTERPRETATION: Artemisinin resistance extends across much of Myanmar. We recorded P falciparum parasites carrying K13-propeller mutations at high prevalence next to the northwestern border with India. Appropriate therapeutic regimens should be tested urgently and implemented comprehensively if spread of artemisinin resistance to other regions is to be avoided. FUNDING: Wellcome Trust-Mahidol University-Oxford Tropical Medicine Research Programme and the Bill & Melinda Gates Foundation.

Degenerate adaptor sequences for detecting PCR duplicates in reduced representation sequencing data improve genotype calling accuracy.

RAD-tag is a powerful tool for high-throughput genotyping. It relies on PCR amplification of the starting material, following enzymatic digestion and sequencing adaptor ligation. Amplification introduces duplicate reads into the data, which arise from the same template molecule and are statistically nonindependent, potentially introducing errors into genotype calling. In shotgun sequencing, data duplicates are removed by filtering reads starting at the same position in the alignment. However, restriction enzymes target specific locations within the genome, causing reads to start in the same place, and making it difficult to estimate the extent of PCR duplication. Here, we introduce a slight change to the Illumina sequencing adaptor chemistry, appending a unique four-base tag to the first index read, which allows duplicate discrimination in aligned data. This approach was validated on the Illumina MiSeq platform, using double-digest libraries of ants (Wasmannia auropunctata) and yeast (Saccharomyces cerevisiae) with known genotypes, producing modest though statistically significant gains in the odds of calling a genotype accurately. More importantly, removing duplicates also corrected for strong sample-to-sample variability of genotype calling accuracy seen in the ant samples. For libraries prepared from low-input degraded museum bird samples (Mixornis gularis), which had low complexity, having been generated from relatively few starting molecules, adaptor tags show that virtually all of the genotypes were called with inflated confidence as a result of PCR duplicates. Quantification of library complexity by adaptor tagging does not significantly increase the difficulty of the overall workflow or its cost, but corrects for differences in quality between samples and permits analysis of low-input material.

Outcomes of chemoradiation for patients with locally advanced non-small-cell lung cancer.

BACKGROUND: Historically, long-term survival rates in locally advanced non-small-cell lung cancer (NSCLC) have been disappointingly low, and treatment toxicities have been significant. AIMS: To assess survival outcomes, treatment toxicities, patterns of disease recurrence and prognostic variables for patients with locally advanced NSCLC treated with concurrent chemoradiation. METHODS: Patients who completed treatment with chemotherapy and simultaneous chest irradiation for locally advanced NSCLC at the Royal Prince Alfred Hospital (Sydney, Australia) in the period January 1994 to July 2009 were identified. We retrospectively reviewed the patients' medical records to obtain patient demographic data, clinical data, information on tumour characteristics and treatment administered, and outcome data such as survival, treatment toxicities and tumour recurrence patterns. RESULTS: Our patient cohort consisted largely of urban-dwelling male smokers with good baseline performance status. As of December 2012, 93/105 patients had died. Median overall and progression-free survival was 20 months and 11 months respectively. The 5-year survival rate was 17%. Eight patients had survived longer than 8 years, and 13 patients enjoyed progression-free survival longer than 3 years. Locoregional tumour recurrence occurred most frequently, followed by brain and bone metastases. Adverse effects from chemoradiation included varying degrees of gastrointestinal, pulmonary and haematological toxicity. Three deaths occurred from radiation-induced pneumonitis. Weight loss at presentation was statistically significantly associated with worse overall survival in univariate analyses (P = 0.01). CONCLUSIONS: Our survival results are consistent with the recent international literature and indicate that a proportion of patients with locally advanced NSCLC can enjoy prolonged survival following treatment with concurrent chemoradiation.

Proviral integration site 2 is required for interleukin-6 expression induced by interleukin-1, tumour necrosis factor-α and lipopolysaccharide.

PIM (proviral integration site) kinases are a distinct class of serine/threonine-specific kinases consisting of PIM1, PIM2 and PIM3. PIM2 is known to function in apoptosis pathways. Expression of PIM2 is highly induced by pro-inflammatory stimuli but the role of PIM2 in the expression of pro-inflammatory cytokines is unclear. In this study, we showed that over-expression of PIM2 in HeLa cells as well as in human umbilical vein endothelial cells enhanced interleukin-1ß (IL-1ß) -induced and tumour necrosis factor-α-induced IL-6 expression, whereas over-expression of a kinase-dead PIM2 mutant had the opposite effect. Studies with small interfering RNA specific to PIM2 further confirmed that IL-6 expression in HeLa cells requires PIM2. To investigate the function of PIM2 further, we generated PIM2-deficient mice. It was found that IL-6 production was significantly decreased from PIM2-deficient spleen cells after stimulation with lipopolysaccharide. Taken together, we demonstrated an important function of PIM2 in controlling the expression of the pro-inflammatory cytokine IL-6. PIM2 inhibitors may be beneficial for IL-6-mediated diseases such as rheumatoid arthritis.

Involvement of GATA3 in protein kinase C theta-induced Th2 cytokine expression.

Protein kinase C theta (PKCtheta) is essential for T cell activation, as it is required for the activation of NF-kappaB and expression of IL-2. PKCtheta has also been shown to affect NFAT activation and Th2 differentiation. To better understand the role of PKCtheta in the regulation of T helper cells, we used PKCtheta-deficient DO11.10 transgenic T cells to study its role in vitro. DO11.10 Th1 cells deficient in PKCtheta produced significantly less TNF-alpha and IL-2. The expression of Th2 cytokines, including IL-4, IL-5, IL-10, IL-13 and IL-24 was significantly reduced in PKCtheta-deficient T cells. Moreover, the expression of the Th2 transcription factor, GATA3, was significantly reduced in PKCtheta-deficient T cells. Overexpression of GATA3 by retroviral infection in PKCtheta-deficient T cells resulted in increased expansion of IL-4-producing T cells and higher IL-4 production than that of wild type Th2 cells. IL-5, IL-10, IL-13 and IL-24 expressions were also rescued by GATA3 overexpression. Our observations suggest that PKCtheta regulates Th2 cytokine expression via GATA3.

The needs of the family of critically ill neurosurgical patients: a comparison of nurses' and family members' perceptions.

In this study, researchers identified the important needs of family members of critically ill neurosurgical patients and explored the relationship between needs and unmet needs as perceived by nurses and family members. A total of 52 family members and 36 nurses in three neurosurgical special care units in Hong Kong were asked to complete the Chinese version of the 45-item Critical Care Family Needs Inventory. The rank order of most important needs reported by family members indicates that the majority of needs are related to assurance; needs for support and comfort were much less important. When rating needs, nurses underrated most of the needs considered important by family members. Needs for proximity were also underrated in importance by nurses when compared to family ratings, and needs for support were heavily overrated by nurses. The needs for proximity were least met. An inverse relationship between nurses' ratings of importance and the frequency of unmet needs was demonstrated. The most important need that was also largely unmet was having a specific person call when unable to visit. The findings of this study indicate areas of unmet need that require additional nursing interventions.

Duplication of the rectum: report of four cases and review of the literature.

Rectal duplications are rare anomalies. Recently, we observed four cases of rectal duplication, each presenting with different clinical features including chronic constipation, a prolapsing rectal "polyp, " a "growth" from the vulva, and acute retention of urine. The variety of clinical presentations may lead to delay in diagnosis and multiple operations.

Thoracoscopy assisted pulmonary lobectomy.

BACKGROUND: This report describes a preliminary experience with six patients undergoing video imaged thoracoscopic pulmonary lobectomy. METHODS: Three left upper lobectomies, and one each of right upper, right lower and left lower lobectomy were undertaken. The resections were performed as orthodox dissectional lobectomy procedures but were carried out under videothoracoscopic imaging with instruments introduced through two stab incisions. The entire resected lobe was delivered through a 7 cm submammary intercostal incision. RESULTS: There were no operative deaths or complications attributable to the technique. In three other patients conversion to an open thoracotomy was required because of bleeding (two cases) or obscure anatomy (one case). Post-operative pain in those undergoing thoracoscopic resection was less than that encountered with standard thoracotomy and early clinic review showed the patients to be pain free with excellent shoulder movement. CONCLUSIONS: Major pulmonary resection according to standard cancer practices is feasible with videothoracoscopic techniques. This approach is likely to offer considerable functional benefit to patients. Specimen delivery through the submammary incision imposes a 5 cm primary lesion size limitation. Detailed mediastinal assessment is necessary to exclude N2 status before undertaking thoracoscopic surgery.

Spectrin breakdown products increase with age in telencephalon of mouse brain.

Calcium activated proteolysis of brain spectrin produces characteristic breakdown products (BDPs), the concentrations of which increase markedly in many instances of brain pathology. Results reported here indicate that levels of the BDPs rise with age (3-30 months) in the telencephalon but not in the hindbrain of Balb/c mice. These observations suggest that spectrin breakdown is a pathologic biochemical marker which increases with age in some but not all brain regions.