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Aim This study aimed to investigate the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on all types of acute cardiac admissions (ACAs) and cardiac mortality in Malta. Methods Number, characteristics and delay to presentation of ACAs to our institution during the study period (28 February–30 April 2020) were compared with the corresponding 2019 period. Non-parametric correlation analyses between daily SARS-CoV-2 cases in Malta, Italy and the UK and daily ACAs were performed. Differences in cardiac death distribution (community vs. in-hospital) during the two periods were analysed. Results There was a significant decline in daily ACAs in 2020 (median 3 [IQR 3]) vs. 2019 (median 5 [IQR 4]), p

Background The effect of ambient temperature on respiratory mortality has been consistently observed throughout the world under different climate change scenarios. Countries experiencing greater inter–annual variability in winter temperatures (and may not be lowest winter temperatures) have greater excess winter mortality compared to countries with colder winters. This study investigates the association between temperature and respiratory deaths in Malta which has one of the highest population densities in the world with a climate that is very hot in summer and mild in winter. Methods Daily number of respiratory deaths (7679 deaths) and meteorological data (daily average temperature, daily average humidity) were obtained from January 1992 to December 2017. The hot and cold effects were estimated at different temperatures using distributed lag non-linear models (DLNM) with a Poisson distribution, controlling for time trend, relative humidity and holidays. The reference temperature (MMT) for the minimum response-exposure relationship was estimated and the harvesting effects of daily temperature (0–27 lag days) were investigated for daily respiratory mortality. Effects were also explored for different age groups, gender and time periods. Results Cooler temperatures (8–15 °C) were significantly related to higher respiratory mortality. At 8.9 °C (1st percentile), the overall effect of daily mean temperature was related to respiratory deaths (RR 2.24, 95%CI 1.10–4.54). These effects were also found for males (95%CI 1.06–7.77) and males across different age groups (Males Over 65 years: RR 4.85, 95%CI 2.02–11.63 vs Males between 16 and 64 years: RR 5.00, 95%CI 2.08–12.03) but not for females. Interestingly, colder temperatures were related to respiratory deaths in the earliest time period (1992–2000), however, no strong cold effect was observed for later periods (2000–2017). In contrast, no heat effect was observed during the study period and across other groups. Conclusions The higher risk for cold-related respiratory mortality observed in this study could be due to greater inter-annual variability in winter temperatures which needs further exploration after adjusting for potential physical and socio-demographic attributes. The study provides useful evidence for policymakers to improve local warning systems, adaptation, and intervention strategies to reduce the impact of cold temperatures.

(1) Objectives: The study aimed to examine data from Malta’s Health Interview Survey (HIS) to assess differences between persons in employment and those retired, across different time periods. (2) Methods: A repeat cross-sectional design was adopted. Data that were collected over a period of 12 years included three cross-sectional HIS waves (2002, 2008 and 2014). Data were analysed cross-sectionally and longitudinally using multilevel analysis. (3) Results: In total, 4690 participants between the ages of 50 and 74 years provided data on physical activity (PA). A statistical difference was found between those employed and retired, with the latter undertaking less PA MET min per week in 2002 and 2008. There was no difference in 2014. When adjusting for covariates, people in employment carried out less PA MET min per week (OR-0.16–−0.02) compared to retired individuals. Using multilevel modelling, this study shows that individual factors such BMI and long-standing illness are predictors of PA behaviour as opposed to time trends. (4) Conclusion: Retirement can increase PA measured in MET minutes per week. Individual factors such as BMI, long-standing health problems and self-rated health could be causing the higher levels seen in the employed population during the studied period.

The coronavirus disease (COVID-19) global pandemic is ongoing, with Europe and the Americas the current epicentres of transmission. As at 1 January, 2021, over 82 million cases and 1.8 million deaths have been reported globally [1,2]. In Europe alone, the number of cases reported has now passed 26 million, with over 580,000 laboratory-confirmed COVID-19 deaths [3,4]. As many COVID-19 cases and deaths may not be confirmed by virological testing, and as the pandemic causes a range of indirect and collateral harms, the officially reported figures of laboratory-confirmed COVID-19 cases and deaths represent only a part of the total disease, mortality and overall public health burden associated with the pandemic. Here we report some noteworthy estimates of a marked increase in excess all-cause mortality in Europe coinciding with a steep second wave of COVID-19 in many countries since September 2020.

Objectives. This study comes at an opportune time due to recent introduction of the National Cervical Cancer Screen- ing programme in Malta. It aims to assess the knowledge of 25-64 year-old females on cervical cancer and attitudes towards screening. Study design. A cross-sectional, telephone-based, quantitative survey conducted in 2017. Methods. The survey tool was based on the Cervical Cancer Awareness Measure questionnaire and was carried out among a random stratified sample of females of 25-64 years, resident in Malta. Multivariate logistic regression models were applied. Results. 407 females (85% response rate) were interviewed. Knowledge of cervical cancer risk factors and symptoms was found to be significantly higher in women with a higher level of education (p < 0.001). Cervical screening was attended every 3 years by 69% of respondents. Regular attendees were more likely to have children (p = 0.001), have experienced cancer in a close family member (p = 0.002), and were between 35-44 and 45-54 years old (p < 0.001). The main reasons for non-attendance were embarrassment, fear of the test and fear of the result. Conclusion. This research provides a better understanding of who are the vulnerable groups with respect to cervical cancer knowledge and screening attendance. Improving health literacy and implementing health promotion campaigns will improve early symptom recognition, risk factor knowledge and attendance for screening., Journal of Preventive Medicine and Hygiene, Vol. 61 No. 4 (2020): 2020614

There is a significant relationship between ambient temperature and mortality. In healthy individuals with no underlying co-morbid conditions, there is an efficient heat regulation system which enables the body to effectively handle thermal stress. However, in vulnerable groups, especially in elderly over the age of 65 years, infants and individuals with co-morbid cardiovascular and/or respiratory conditions, there is a deficiency in thermoregulation. When temperatures exceed a certain limit, being cold winter spells or heat waves, there is an increase in the number of deaths. In particular, it has been shown that at temperatures above 27 °C, the daily mortality rate increases more rapidly per degree rise compared to when it drops below 27 °C. This is especially of relevance with the current emergency of global warming. Besides the direct effect of temperature rises on human health, global warming will have a negative impact on primary producers and livestock, leading to malnutrition, which will in turn lead to a myriad of health related issues. This is further exacerbated by environmental pollution. Public health measures that countries should follow should include not only health-related information strategies aiming to reduce the exposure to heat for vulnerable individuals and the community, but improved urban planning and reduction in energy consumption, among many others. This will reduce the carbon footprint and help avert global warming, thus reducing mortality.

Background: Since 2015, the World Health Organization (WHO) has recommended a commercially available lateral-flow urine LAM test (Alere-LAM) to assist in the diagnosis of tuberculosis (TB) in severely ill people living with HIV (PLHIV). The test can rapidly detect TB in severely ill PLHIV and can identify PLHIV most at-risk of death, leading to mortality reductions. However, its uptake in countries with high burdens of TB and HIV has been slow. To assess the current use landscape and identify barriers to the adoption of Alere-LAM, we conducted a questionnaire-based study in 31 high TB and HIV/AIDS burden countries. Methods: Between November 2018 and December 2019, we collected responses to a semi-structured questionnaire that had been emailed to staff and affiliates of National TB Programs or HIV/AIDS Programs, Ministries of Health, and TB or HIV institutes of 31 high TB/HIV burden countries. Questions concerned country policies, adoption, and current use of Alere-LAM testing, as well as testing algorithms and barriers preventing Alere-LAM uptake. Results: We received questionnaire responses from 24 out of 31 (77%) high TB/HIV burden countries. Of these 24 countries, 11 (46%) had adopted Alere-LAM policies, with only five (21%) countries currently using Alere-LAM testing. Testing algorithms were generally aligned with WHO recommendations. Fifteen countries (63%) said they were planning to implement Alere-LAM testing in the near future. The most commonly cited constraint to adoption and implementation was budget limitations. Additional barriers to Alere-LAM implementation included lack of country-specific data and piloting, administrative hurdles such as regulatory agency approval, lack of coordination between National TB and HIV programs, and small perceived patient population. Conclusion: Responses to our questionnaire demonstrate the persistent gap between country-level policy and real-world use of Alere-LAM, as well as specific barriers that must be addressed to scale-up testing in PLHIV.

Generalized estimating equation (GEE) models are extensions of generalized linear models by relaxing the assumption of independence. These models are appropriate to analyze correlated longitudinal responses that follow any distribution that is a member of the exponential family. This model is used to relate daily mortality rate of Maltese adults aged 65 years and over with a number of predictors, including apparent temperature, season, and year. The GEE model identifies a number of significant main and interaction effects on daily mortality rates and shows that mortality rate and temperature are related by a quadratic function.

Background Interferon-gamma (IFN-γ) release assays (IGRAs) are used to diagnose tuberculosis (TB) but not to measure treatment response. Objective To measure IFN-γ response to active anti-tuberculosis treatment. Design Patients from the Henan Provincial Chest Hospital, Henan, China, with TB symptoms and/or signs were enrolled into this prospective, observational cohort study and followed for 6 months of treatment, with blood and sputum samples collected at 0, 2, 4, 6, 8, 16 and 24 weeks. The QuantiFERON® TB-Gold assay was run on collected blood samples. Participants received a follow-up telephone call at 24 months to determine relapse status. Results Of the 152 TB patients enrolled, 135 were eligible for this analysis: 118 pulmonary (PTB) and 17 extra-pulmonary TB (EPTB) patients. IFN-γ levels declined significantly over time among all patients (P = 0.002), with this decline driven by PTB patients (P = 0.001), largely during the initial 8 weeks of treatment (P = 0.019). IFN-γ levels did not change among EPTB patients over time or against baseline culture or drug resistance status. Conclusion After 6 months of effective anti-tuberculosis treatment, IFN-γ levels decreased significantly in PTB patients, largely over the initial 8 weeks of treatment. IFN-γ concentrations may offer some value for monitoring anti-tuberculosis treatment response among PTB patients.

Objectives: Weekly monitoring of European all-cause excess mortality, the EuroMOMO network, observed high excess mortality during the influenza B/Yamagata dominated 2017/18 winter season, especially among elderly. We describe all-cause excess and influenza-attributable mortality during the season 2017/18 in Europe. Methods: Based on weekly reporting of mortality from 24 European countries or sub-national regions, representing 60% of the European population excluding the Russian and Turkish parts of Europe, we estimated age stratified all-cause excess morality using the EuroMOMO model. In addition, age stratified all-cause influenza-attributable mortality was estimated using the FluMOMO algorithm, incorporating influenza activity based on clinical and virological surveillance data, and adjusting for extreme temperatures. Results: Excess mortality was mainly attributable to influenza activity from December 2017 to April 2018, but also due to exceptionally low temperatures in February-March 2018. The pattern and extent of mortality excess was similar to the previous A(H3N2) dominated seasons, 2014/15 and 2016/17. The 2017/18 overall all-cause influenza-attributable mortality was estimated to be 25.4 (95%CI 25.0-25.8) per 100,000 population; 118.2 (116.4-119.9) for persons aged 65. Extending to the European population this translates into over-all 152,000 deaths. Conclusions: The high mortality among elderly was unexpected in an influenza B dominated season, which commonly are considered to cause mild illness, mainly among children. Even though A(H3N2) also circulated in the 2017/18 season and may have contributed to the excess mortality among the elderly, the common perception of influenza B only having a modest impact on excess mortality in the older population may need to be reconsidered. info:eu-repo/semantics/publishedVersion

Shortening the lengthy treatment duration for tuberculosis patients is a major goal of current drug development efforts. The common marmoset develops human-like disease pathology and offers an attractive model to better understand the basis for relapse and test regimens for effective shorter duration therapy. We treated Mycobacterium tuberculosis -infected marmosets with two drug regimens known to differ in their relapse rates in human clinical trials: the standard four-drug combination of isoniazid, rifampin, pyrazinamide, and ethambutol (HRZE) that has very low relapse rates and the combination of isoniazid and streptomycin that is associated with higher relapse rates. As early as 2 weeks, the more sterilizing regimen significantly reduced the volume of lung disease by computed tomography ( P = 0.035) and also significantly reduced uptake of [ 18 F]-2-fluoro-2-deoxyglucose by positron emission tomography ( P = 0.049). After 6 weeks of therapy, both treatments caused similar reductions in granuloma bacterial load, but the more sterilizing, four-drug regimen caused greater reduction in bacterial load in cavitary lesions ( P = 0.009). These findings, combined with the association in humans between cavitary disease and relapse, suggest that the basis for improved sterilizing activity of the four-drug combination is both its faster disease volume resolution and its stronger sterilizing effect on cavitary lesions. Definitive data from relapse experiments are needed to support this observation.

Background Neonatal mortality is a public health concern, and congenital anomalies contribute significantly to this mortality. This paper describes trends in neonatal mortality in Malta separately for congenital anomaly and non-congenital anomaly causes. Methods Data for neonatal deaths of 22-week gestation onwards registered between 1994–2013 were obtained from the National Mortality Register. Chi-square tests were used to analyse 5-year time trends and differences in proportions of causes of neonatal deaths. Neonatal mortality was compared with other European countries. Results Between 1994 and 2013, 441 neonatal deaths and 84 821 livebirths were registered, giving a neonatal mortality of 5.2 per 1000 livebirths. Congenital anomalies accounted for 36.7% (n = 162) of the neonatal deaths, while the remaining 63.3% (n = 279) were attributed to non-congenital causes. During the 20-year period, neonatal mortality due to non-congenital causes decreased from 4.6 per 1000 livebirths in 1994–98 to 2.5 per 1000 in 2009–13, while that due to congenital anomalies remained stable (2.0 per 1000 livebirths in 1994–98 and 2.2 per 1000 in 2009–13). This has resulted in comparatively higher proportions of neonatal deaths attributed to congenital anomalies in recent years (45.9% in 2009–13 vs. 29.9% in 1994–98). Comparing neonatal mortality reported from European countries, Malta has a high rate most marked for deaths due to congenital anomalies. Conclusions During 1994–2013, neonatal mortality has decreased due to a decline of non-congenital causes of death, possibly related to improved health care. The proportionate neonatal mortality attributed to congenital anomalies has increased and is the highest reported from Europe. This may be explained by the fact that termination of pregnancy is illegal in Malta.

Tuberculosis (TB) causes almost 2 million deaths annually, and an increasing number of patients are resistant to existing therapies. Patients who have TB require lengthy chemotherapy, possibly because of poor penetration of antibiotics into granulomas where the bacilli reside. Granulomas are morphologically similar to solid cancerous tumors in that they contain hypoxic microenvironments and can be highly fibrotic. Here, we show that TB-infected rabbits have impaired small molecule distribution into these disease sites due to a functionally abnormal vasculature, with a low-molecular-weight tracer accumulating only in peripheral regions of granulomatous lesions. Granuloma-associated vessels are morphologically and spatially heterogeneous, with poor vessel pericyte coverage in both human and experimental rabbit TB granulomas. Moreover, we found enhanced VEGF expression in both species. In tumors, antiangiogenic, specifically anti-VEGF, treatments can "normalize" their vasculature, reducing hypoxia and creating a window of opportunity for concurrent chemotherapy; thus, we investigated vessel normalization in rabbit TB granulomas. Treatment of TB-infected rabbits with the anti-VEGF antibody bevacizumab significantly decreased the total number of vessels while normalizing those vessels that remained. As a result, hypoxic fractions of these granulomas were reduced and small molecule tracer delivery was increased. These findings demonstrate that bevacizumab treatment promotes vascular normalization, improves small molecule delivery, and decreases hypoxia in TB granulomas, thereby providing a potential avenue to improve delivery and efficacy of current treatment regimens.

Demographic trends in Europe are currently being shaped by an ageing population, falling fertility rates and diverse migration flows. Fertility rates are lowest in Eastern and Southern Europe with Eastern Europe also experiencing the lowest net migration and an exodus of its working population. All regions in Europe are experiencing aging of their population with some countries having the added burden of high rates of unemployment among the working age population. The impact of these demographic changes on the current and future public health of the country depends on how countries have been preparing and adapting to demographic changes over the past years. Changes in age structure and ethnic composition will put further strain on health care and welfare systems and requires careful planning. A multi-faceted approach which goes beyond the health care system is required and countries need to look beyond their borders in search as to how countries are tackling these important issues. As Europe ages the concept of healthy aging should become an increasing priority focus for European Public Health.

Since December 2016, excess all-cause mortality was observed in many European countries, especially among people aged ≥ 65 years. We estimated all-cause and influenza-attributable mortality in 19 European countries/regions. Excess mortality was primarily explained by circulation of influenza virus A(H3N2). Cold weather snaps contributed in some countries. The pattern was similar to the last major influenza A(H3N2) season in 2014/15 in Europe, although starting earlier in line with the early influenza season start. info:eu-repo/semantics/publishedVersion

The carbapenems imipenem and meropenem in combination with clavulanic acid reduced the bacterial burden in Mycobacterium tuberculosis -infected macrophages by 2 logs over 6 days. Despite poor stability in solution and a short half-life in rodents, treatment of chronically infected mice revealed significant reductions of bacterial burden in the lungs and spleens. Our results show that meropenem has activity in two in vivo systems, but stability and pharmacokinetics of long-term administration will offer significant challenges to clinical evaluation.

As an initial step in developing novel antibacterials against Burkholderia pseudomallei, we have characterized the FabI enoyl-ACP reductase homologues in the type II fatty acid biosynthesis pathway from this organism and performed an initial enzyme inhibition study.A BLAST analysis identified two FabI enoyl-ACP reductase homologues, bpmFabI-1 and bpmFabI-2, in the B. pseudomallei genome, which were cloned, overexpressed in Escherichia coli and purified. Steady-state kinetics was used to determine the reaction mechanism and the sensitivity of bpmFabI-1 to four diphenyl ether FabI inhibitors. The antibacterial activity of the inhibitors was assessed using a wild-type strain of Burkholderia thailandensis (E264) and an efflux pump mutant (Bt38).Consistent with its annotation as an enoyl-ACP reductase, bpmFabI-1 catalysed the NADH-dependent reduction of 2-trans-dodecenoyl-CoA via a sequential Bi Bi mechanism. In contrast, bpmFabI-2 was inactive with all substrates tested and only bpmfabI-1 was transcriptionally active under the growth conditions employed. The sensitivity of bpmFabI-1 to four diphenyl ethers was evaluated and in each case the compounds were slow-onset inhibitors with K(i) values of 0.5-2 nM. In addition, triclosan and PT01 had MIC values of 30 and 70 mg/L for B. pseudomallei as well as a wild-type strain of B. thailandensis (E264), but MIC values of1 mg/L for the efflux pump mutant Bt38. A reduction in MIC values was also observed for the pump mutant strain with the other diphenyl ethers.Provided that efflux can be circumvented, bpmFabI-1 is a suitable target for drug discovery.

Objectives: The National Institute of Allergy and Infectious Disease classifies Francisella tularensis as a Category A priority pathogen. Despite the availability of drugs for treating tularaemia, the mortality in naturally acquired cases can still approach 30%. In addition, the usefulness of existing drugs for treatment in response to exposure or for prophylaxis is limited because of toxicity and delivery concerns. The aim of this study was to assess the efficacy of the lead alkyl-substituted diphenyl ether, SBPT04, in the F. tularensis murine model of infection. Methods: SBPT04 was delivered by intraperitoneal (ip) and oral (po) routes, and mice were monitored for morbidity, mortality and relapse of disease. Pharmacokinetic studies were performed to evaluate bioavailability. Phase I and Phase II metabolism of SBPT04 was assessed in mouse and human microsomes. Results: SBPT04, a potent inhibitor of the enoyl-ACP reductase enzyme ftuFabI, has efficacy against F. tularensis in the murine model of infection when delivered by both ip and po routes. SBPT04 delivered ip cleared infection by day 4 of treatment, and SBPT04 delivered po resulted in delayed dissemination. Importantly, SBPT04 delivered ip or po demonstrated efficacy with no signs of relapse of disease. Pharmacokinetic studies show increased serum concentrations following ip delivery compared with po delivery, which correlates with the observed survival rate of 100%. Conclusions: In addition to being a potent lead, this work substantiates substituted diphenyl ethers as a platform for the development of novel broad-spectrum chemotherapeutics to other bacterial agents in addition to F. tularensis.

Francisella tularensis is a highly virulent and contagious Gram-negative intracellular bacterium that causes the disease tularemia in mammals. The high infectivity and the ability of the bacterium to survive for weeks in a cool, moist environment have raised the possibility that this organism could be exploited deliberately as a potential biological weapon. Fatty acid biosynthesis (FAS-II) is essential for bacterial viability and has been validated as a target for the discovery of novel antibacterials. The FAS-II enoyl reductase ftuFabI has been cloned and expressed, and a series of diphenyl ethers have been identified that are subnanomolar inhibitors of the enzyme with MIC90 values as low as 0.00018 microg mL(-1). The existence of a linear correlation between the Ki and MIC values strongly suggests that the antibacterial activity of the diphenyl ethers results from direct inhibition of ftuFabI within the cell. The compounds are slow-onset inhibitors of ftuFabI, and the residence time of the inhibitors on the enzyme correlates with their in vivo activity in a mouse model of tularemia infection. Significantly, the rate of breakdown of the enzyme-inhibitor complex is a better predictor of in vivo activity than the overall thermodynamic stability of the complex, a concept that has important implications for the discovery of novel chemotherapeutics that normally rely on equilibrium measurements of potency.

This study aimed to, prospectively, over the 5-year period 2000–2004, accurately determine features of cutaneous melanoma in the Maltese Islands. Data from clinicians were supplemented by histology reports, and where necessary, histology slides were reviewed. Information collected included demographic details including age and gender, anatomical site, Clark’s level, Breslow thickness and clinico-pathological melanoma type. During the study period the age-standardised (European Standard Population) rates for invasive melanoma were 8.81 per 100,000 (males) and 7.29 per 100,000 (females) and increased with age. By the end of the study, information on 166 cases of primary invasive cutaneous malignant melanoma were collected. The commonest site affected in males was the trunk (54%) and in females the lower limbs (41%). Overall, 33.8% of invasive melanomas had a Breslow thickness >1.5 mm. The initial melanoma excision was performed by a dermatologist in 68.2% and plastic surgeon in 20.8%. More cases presented in late spring and summer, particularly in females. Melanoma incidence in Malta is lower than that in high-incidence countries and northern Europe and is similar to that in southern Europe. However, incidence appears to be increasing and a relatively high proportion of patients present with thick lesions emphasising the importance of continued efforts to diagnose cases earlier.

Stress-induced molecular programs designed to stall division progression are nearly ubiquitous in bacteria, with one well-known example being the participation of the SulA septum inhibiting protein in the SOS DNA damage repair response. Mycobacteria similarly demonstrate stress-altered growth kinetics, however no such regulators have been found in these organisms. We therefore set out to identify SulA-like regulatory proteins in Mycobacterium tuberculosis. A bioinformatics modeling-based approach led to the identification of rv2216 as encoding for a protein with weak similarity to SulA, further analysis distinguished this protein as belonging to a group of previously uncharacterized growth promoting proteins. We have named the mycobacterial protein encoded by rv2216 morphology altering division regulator protein 1, MadR1. Overexpression of madR1 modulated cell length while maintaining growth kinetics similar to wild-type, and increased the proportion of bent or V-form cells in the population. The presence of MadR1-GFP at regions of cellular elongation (poles) and morphological differentiation (V-form) suggests MadR1 involvement in phenotypic herterogeneity and longitudinal cellular growth. Global transcriptional analysis indicated that MadR1 functionality is linked to lipid editing programs required for growth and persistence. This is the first report to differentiate the larger class of these conserved proteins from SulA proteins and characterizes MadR1 effects on the mycobacterial cell.

The development of effective chemotherapy for the treatment of tuberculosis (TB) began in the 1940s and has been reinvigorated recently due to concern regarding the emergence of highly drug-resistant TB strains. This chapter explores the medicinal chemistry efforts that gave rise to current frontline and second-line drugs in global use today and attempts to comprehensively summarize ongoing discovery and lead optimization programs being conducted in both the private and the public sector. TB has a large number of disease-specific considerations and constraints that introduce significant complexity in drug discovery efforts. Conceptually, the disease encompasses all the drug discovery challenges of both infectious diseases and oncology, and integrating these considerations into programs that often demand collaboration between industry and academia is both challenging and rewarding.

Tuberculosis (TB) causes almost 2 million deaths annually, and an increasing number of patients are resistant to existing therapies. TB patients require lengthy chemotherapy, possibly because of poor penetration of antibiotics into granulomas where the bacilli reside. Granulomas are morphologically similar to solid cancerous tumors in that they contain hypoxic microenvironments and can be highly fibrotic. Here we show that TB-infected rabbits have impaired small molecule distribution into these disease sites due to a functionally abnormal vasculature, with a low molecular weight tracer accumulating only in peripheral regions of granulomatous lesions. Granuloma-associated vessels are morphologically and spatially heterogeneous, with poor vessel pericyte coverage in both human and experimental rabbit TB granulomas. Moreover, we found enhanced vascular endothelial growth factor (VEGF) expression in both species. In tumors, anti-angiogenic, specifically anti-VEGF, treatments can “normalize” their vasculature, reducing hypoxia and creating a window-of-opportunity for conjunctive chemotherapy; thus, we investigated vessel normalization in rabbit TB granulomas. Treatment of TB-infected rabbits with the anti-VEGF antibody bevacizumab significantly decreased the total number of vessels while normalizing those that remained. As a result, hypoxic fractions of these granulomas were reduced and small molecule tracer delivery increased. These findings demonstrate that bevacizumab treatment promotes vascular normalization, improves small molecule delivery, and decreases hypoxia in TB granulomas, thereby providing a potential new avenue to improve delivery and efficacy of current treatment regimens. Citation Format: Meenal Datta, Laura E. Via, Walid S. Kamoun, Chong Liu, Wei Chen, Giorgio Seano, Danielle M. Weiner, Daniel Schimel, Kathleen England, John D. Martin, Xing Gao, Lei Xu, Clifton E. Barry, III, Rakesh K. Jain. Anti-VEGF treatment normalizes tuberculosis granuloma vasculature and improves small molecule delivery. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B19.

Background Proteins that are involved in regulation of cell division and cell cycle progression remain undefined in Mycobacterium tuberculosis. In addition, there is a growing appreciation that regulation of cell replication at the point of division is important in establishing a non-replicating persistent state. Accordingly, the objective of this study was to use a systematic approach consisting of consensus-modeling bioinformatics, ultrastructural analysis, and transcriptional mapping to identify septum regulatory proteins that participate in adaptive metabolic responses in M. tuberculosis. Results Septum site determining protein (Ssd), encoded by rv3660c was discovered to be an ortholog of septum site regulating proteins in actinobacteria by bioinformatics analysis. Increased expression of ssd in M. smegmatis and M. tuberculosis inhibited septum formation resulting in elongated cells devoid of septa. Transcriptional mapping in M. tuberculosis showed that increased ssd expression elicited a unique response including the dormancy regulon and alternative sigma factors that are thought to play a role in adaptive metabolism. Disruption of rv3660c by transposon insertion negated the unique transcriptional response and led to a reduced bacterial length. Conclusions This study establishes the first connection between a septum regulatory protein and induction of alternative metabolism consisting of alternative sigma factors and the dormancy regulon that is associated with establishing a non-replicating persistent intracellular lifestyle. The identification of a regulatory component involved in cell cycle regulation linked to the dormancy response, whether directly or indirectly, provides a foundation for additional studies and furthers our understanding of the complex mechanisms involved in establishing a non-replicating state and resumption of growth.

Background Molecular programs employed by Mycobacterium tuberculosis (Mtb) for the establishment of non-replicating persistence (NRP) are poorly understood. In order to investigate mechanisms regulating entry into NRP, we asked how cell cycle regulation is linked to downstream adaptations that ultimately result in NRP. Based on previous reports and our recent studies, we reason that, in order to establish NRP, cells are halted in the cell cycle at the point of septum formation by coupled regulatory mechanisms. Results Using bioinformatic consensus modeling, we identified an alternative cell cycle regulatory element, SojMtb encoded by rv1708. SojMtb coordinates a regulatory mechanism involving cell cycle control at the point of septum formation and elicits the induction of the MazF6 toxin. MazF6 functions as an mRNA interferase leading to bacteriostasis that can be prevented by interaction with its cognate antitoxin, MazE6. Further, MazEF6 acts independently of other Maz family toxin:antitoxin pairs. Notably, soj Mtb and mazEF6 transcripts where identified at 20, 40 and 100 days post-infection in increasing abundance indicating a role in adaption during chronic infection. Conclusions Here we present the first evidence of a coupled regulatory system in which cell cycle regulation via SojMtb is linked to downstream adaptations that are facilitated through the activity of the MazEF6 TA pair.