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The disaccharide trehalose is essential for viability of Mycobacterium tuberculosis , which synthesizes trehalose de novo but can also utilize exogenous trehalose. The mycobacterial cell wall encompasses two permeability barriers, the cytoplasmic membrane and the outer mycolic acid-containing mycomembrane. The ABC transporter LpqY–SugA–SugB–SugC has previously been demonstrated to mediate the specific uptake of trehalose across the cytoplasmic membrane. However, it is still unclear how the transport of trehalose molecules across the mycomembrane is mediated. In this study, we harnessed the antimycobacterial activity of the analogue 6-azido trehalose to select for spontaneous resistant M. tuberculosis mutants in a merodiploid strain harbouring two LpqY–SugA–SugB–SugC copies. Mutations mediating resistance to 6-azido trehalose mapped to the proline–proline–glutamate (PPE) family member PPE51 (Rv3136), which has recently been shown to be an integral mycomembrane protein involved in uptake of low-molecular weight compounds. A site-specific ppe51 gene deletion mutant of M. tuberculosis was unable to grow on trehalose as the sole carbon source. Furthermore, bioorthogonal labelling of the M. tuberculosis Δ ppe51 mutant incubated with 6-azido trehalose corroborated the impaired internalization. Taken together, the results indicate that the transport of trehalose and trehalose analogues across the mycomembrane of M. tuberculosis is exclusively mediated by PPE51.

The obligate intracellular bacterium Chlamydia trachomatis inserts a family of inclusion membrane (Inc) proteins into the membrane of its vacuole (the inclusion). The Inc CpoS is a critical suppressor of host cellular immune surveillance, but the underlying mechanism remained elusive. By complementing a cpoS mutant with various natural orthologs and variants of CpoS, we linked distinct molecular interactions of CpoS to distinct functions. Unexpectedly, we found CpoS to be essential for the formation of inclusion membrane microdomains that control the spatial organization of multiple Incs involved in signaling and modulation of the host cellular cytoskeleton. While the function of CpoS in microdomains was uncoupled from its role in the suppression of host cellular defenses, we found the ability of CpoS to interact with Rab GTPases to be required not only for the manipulation of membrane trafficking, such as to mediate transport of ceramide-derived lipids (sphingolipids) to the inclusion, but also for the inhibition of Stimulator of interferon genes (STING)-dependent type I interferon responses. Indeed, depletion of Rab35 phenocopied the exacerbated interferon responses observed during infection with CpoS-deficient mutants. Overall, our findings highlight the role of Inc–Inc interactions in shaping the inclusion microenvironment and the modulation of membrane trafficking as a pathogenic immune evasion strategy. Chlamydia trachomatis is a prevalent bacterial pathogen that causes blinding ocular scarring and urogenital infections that can lead to infertility and pregnancy complications. Because Chlamydia can only grow within its host cell, boosting the intrinsic defenses of human cells may represent a novel strategy to fight pathogen replication and survival. Hence, CpoS, a Chlamydia protein known to block host cellular defenses, or processes regulated by CpoS, could provide new opportunities for therapeutic intervention. By revealing CpoS as a multifunctional virulence factor and by linking its ability to block host cellular immune signaling to the modulation of membrane trafficking, the present work may provide a foundation for such rationale targeting and advances our understanding of how intracellular bacteria can shape and protect their growth niche.

A mechanistic understanding of how intracellular pathogens evade the intrinsic defenses of their host cells could open up intriguing therapeutic opportunities. Here, we applied a genome-wide genetic screening approach to investigate the nature of the defensive host cell death response suppressed by the membrane trafficking modulator CpoS, an effector protein secreted by the obligate intracellular bacterial pathogen Chlamydia trachomatis . Initially, this work revealed a CpoS-deficient mutant to exhibit a markedly increased dependence on host cellular synthesis of ceramides, the precursors of complex sphingolipids. Using novel microscopic reporters, we then established CpoS’ role in defense evasion to occur by preserving the integrity of Chlamydia ’s parasitophorous vacuole (the inclusion) via ensuring an adequate sphingolipid supply. More specifically, we observed CpoS deficiency to destabilize inclusions, initially characterized by a release of individual bacteria into the host cell cytosol, then followed by inclusion rupture concomitant with host cell death. Exogenous addition of sphingosine stabilized CpoS-deficient inclusions, whereas disruption of host cellular ceramide synthesis destabilized wild-type inclusions. In combination, CpoS deficiency and impaired ceramide synthesis – presumably disrupting both Chlamydia ’s vesicular and non-vesicular sphingolipid supply routes – destabilized inclusions even earlier, resulting in infection clearance and host cell survival rather than host cell death. Overall, this study highlights how the vacuolar pathogen C. trachomatis maintains vacuole integrity by ensuring a steady sphingolipid supply, potentially offering inspiration and directions for future therapeutic strategies targeting parasitophorous vacuoles.

The evolutionarily conserved Hippo signaling pathway regulates organ size and tissue homeostasis. Yes-associated protein (YAP) functions as a transcriptional co-activator and is a critical downstream effector of the Hippo signaling pathway. Altered crosstalk with oncogenic signaling pathways contributes to YAP dysregulation in cancer. Kinase Suppressor of Ras 1 (KSR1) scaffolds the Ras cascade. Some of the functions of the Ras and Hippo pathways in regulating cellular processes are similar. Nevertheless, the potential intersection of Ras and Hippo signaling has not been explored. Here, we identify KSR1 as a previously unrecognized scaffold of the Hippo pathway. We demonstrate that KSR1 constitutively binds to YAP and MST1 and forms a complex with LATS1. Moreover, KSR1 modulates YAP protein levels and its transcriptional activity, at least in part through the RhoA/actin axis. Our findings provide insight into the role of KSR1 as a scaffold of the Hippo signaling that could yield novel therapeutics. KSR1 interacts with and regulates YAP co-transcriptional function, revealing an additional mechanism for growth control and tumourigenesis.

Abstract Disclosure: H. Babu: None. A. Nawaz: None. A.J. Spiro: None. T.D. Hoang: None. M.K. Shakir: None. The overnight 1 mg dexamethasone suppression test (DST) is widely used to screen for Cushing’s Syndrome (CS), especially due to its low incidence of false negative results. Serum cortisol concentrations <1.8 µg/dL suggests adequate HPA axis suppression and usually excludes CS. Concentrations >1.8 µg/dL should be verified with a second test. However, the lack of suppression on DST has been documented previously in 1.46% to 15% of obese patients without CS. Previous studies have suggested simultaneous measurements of serum cortisol, ACTH, and dexamethasone levels after DST to ensure adequate suppression. In this retrospective analysis, we compared the plasma levels of ACTH, dexamethasone, and cortisol levels after 1 mg and 2 mg DST in healthy obese (class III) subjects. The study involved 6 subjects (2 males, 4 females, age 37.5 ± 8.79 years, BMI 43.65 ± 3.12 (kg/m2) who were referred for evaluation of abnormal weight gain. Exclusion criteria for these subjects included diabetes mellitus, pregnancy, history of CS, psychiatric disorders, chronic liver disease, chronic renal failure, malabsorption disorders and nephrotic syndrome. The exclusion of CS was done by history and physical examination showing the lack of specific clinical features of CS. Additionally, none of the subjects were taking drugs that interfered with CYP3A4 or oral contraceptives within 3 months preceding the study. None of the participants reported alcohol consumption of more than 30 g per day. Screening tests for CS involved 1 mg, 2 mg DST, LNSC and 24-hour urine cortisol levels. Results: Following 1 mg DST in 5 subjects, the mean serum cortisol levels were 1.35 ± 0.13 mcg/dL and the mean serum dexamethasone levels, and ACTH levels were 282 ± 44 ng/dL and 5.92 ± 1.56 pg/mL, respectively. However, in one subject (BMI 43.5) the post-DST cortisol level was 2.96 mcg/dL with corresponding dexamethasone level of 138 ng/dL and ACTH level of 21 pg/mL. However, following 2 mg DST in all 6 subjects the serum cortisol levels were less than 1.8 mcg/dL (1.21± 0.22 mcg/dL) with ACTH levels (3.87 ± 0.70 pg/mL) and dexamethasone levels of 482 ± 80 ng/dL. The LNSC levels were normal in 4 subjects (0.010 ± 0.005 mcg/dL), whereas in 2 subjects these were minimally elevated. 24-hour urine cortisol levels were normal in 5 subjects (29.4 ± 3.74 mcg) and in one subject this was mildly elevated (67 mcg). Long term follow-up of all subjects confirmed normal cortisol status. Previous studies have demonstrated that routine CS screening of patients affected by severe obesity is not indicated. However, other investigations have shown CS may occur in a small percentage of severe obese patients. In conclusion, 2 mg DST is a preferred test in obese patients suspected to have CS although 1 mg DST generally can be used as an initial screening test. Additionally, serum dexamethasone and ACTH measurements may still be useful in patients who are suspected to have a false-positive DST. Presentation: 6/1/2024

Abstract Disclosure: H.M. Babu: None. I. Ebrahim: None. N.O. Vietor: None. T.D. Hoang: None. M.K. Shakir: None. Acromegaly usually presents with classic clinical features such as acral enlargement, facial features, headaches, fatigue, hyperhidrosis, snoring and oily skin. However, cases with atypical clinical features have been reported on silent GH-secreting pituitary tumors. The term ‘subclinical acromegaly’ is used to describe patients with GH hypersecretion without clinical features. We are reporting the long-term follow up of 2 patients with silent acromegaly. Case 1: A 40-year-old Caucasian female was seen 6 years ago with symptoms of arthralgias, cognitive slowing, headaches, and depression. Physical examination was completely normal without evidence to suggest acromegaly. While she was undergoing evaluation for multiple sclerosis, a brain MRI revealed a cystic lesion measuring 1.6 x 0.9 cm. An insulin like growth factor- 1 (IGF-1) level was incidentally screened and was elevated at 823 ng/mL (ref 69-227) and a glucose tolerance test (GTT) reported a growth hormone (GH) level of 7.5 ng/mL at its nadir. The patient underwent transsphenoidal surgery and histology of the resected tumor confirmed a somatotroph tumor. Following surgery, IGF-1 level remained mildly elevated (321 ng/mL) and patient has continued to have arthralgias and diaphoresis. She was treated with lanreotide injection which improved her symptoms. A recent MRI revealed no evidence of residual or recurrent tumor along with normal IGF-1 levels. Case 2: A 78-year-old female was admitted to the hospital 9 years ago with bipolar disorder. An MRI of the brain revealed a left pituitary macroadenoma of 1.5 cm. Patient had no clinical features of acromegaly and laboratory studies revealed an IGF-1 level of 768 ng/mL along with a GH level of 8.9 ng/mL at its nadir during a GTT. Serum prolactin level, thyroid functions and adrenal functions were normal. However, patient also had laboratory values consistent with primary hyperparathyroidism. A genetic screen for MEN-I was negative. Although patient agreed to undergo parathyroid surgery, she did not consent for pituitary surgery. Following parathyroid surgery patient remained eucalcemic. Patient was started on octreotide LAR 30 mg every month and she tolerated the treatment well. She did not manifest any acromegalic features during the 9 years of follow up. A recent IGF-1 level was 161 ng/mL and an MRI showed a stable pituitary adenoma (1.6 cm). In conclusion, the diagnosis of acromegaly may be delayed in patients with subclinical acromegaly. Often an incidental finding of a of a pituitary lesion along with an elevated serum IGF-1 level will lead to a diagnosis of subclinical acromegaly. Although pituitary surgery is the preferred choice in these patients, somatostatin analogue treatment may also be effective in selected patients. Finally, our first case emphasizes the importance of a detailed hormonal evaluation in patients who present with cystic pituitary incidentalomas. Presentation: 6/1/2024

The reuse of domestic and industrial wastewater in urban settings of the developing world may harm the health of people through direct contact or via contaminated urban agricultural products and drinking water. We assessed chemical and microbial pollutants in 23 sentinel sites along the wastewater and faecal sludge management and reuse chain of Kampala, Uganda. Water samples were examined for bacteria (thermotolerant coliforms (TTCs), Escherichia coli and Salmonella spp.) and helminth eggs. Physico-chemical parameters were determined. Water, sediment and soil samples and edible plants (yams and sugar cane) were tested for heavy metals. Water samples derived from the Nakivubo wetland showed mean concentrations of TTCs of 2.9 × 10 5  colony-forming units (CFU)/100 mL. Mean E. coli was 9.9 × 10 4  CFU/100 mL. Hookworm eggs were found in 13.5 % of the water samples. Mean concentrations of iron (Fe), copper (Cu) and cadmium (Cd) were 21.5, 3.3 and 0.14 mg/L, respectively. In soil samples, we found a mean lead (Pb) concentration of 132.7 mg/L. In yams, concentrations of Cd, chromium (Cr) and Pb were 4.4, 4.0 and 0.2 mg/L, while the respective concentrations in sugar cane were 8.4, 4.3 and 0.2 mg/L. TTCs and E. coli in the water, Pb in soil, and Cd, Cr and Pb in the plants were above national thresholds. We conclude that there is considerable environmental pollution in the Nakivubo wetland and the Lake Victoria ecosystem in Kampala. Our findings have important public health implications, and we suggest that a system of sentinel surveillance is being implemented that, in turn, can guide adequate responses.

Reuse of wastewater in agriculture is a common feature in the developing world. While this strategy might contribute to the livelihood of farming communities, there are health risks associated with the management and reuse of wastewater and faecal sludge. We visualise here an assessment of health risks along the major wastewater channel in Kampala, Uganda. The visualization brings to bear the context of wastewater reuse activities in the Nakivubo wetlands and emphasises interconnections to disease transmission pathways. The contextual features are complemented with findings from environmental sampling and a cross-sectional epidemiological survey in selected exposure groups. Our documentation can serve as a case study for a step-by-step implementation of risk assessment and management as described in the World Health Organization's 2006 guidelines for the safe use of wastewater, greywater and excreta in light of the forthcoming sanitation safety planning approach.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by widespread areas of abnormal bone formation in muscles, ligaments, tendons and joint capsules. Typically, the symptoms begin in the first decade of life with episodes of painful inflammatory soft tissue swellings. Gradually, there occurs restriction of motion at various joints, severely limiting the activities of daily living and the quality of life of such patients by the third decade of life. There is no definite cure available for the disease and the current treatment options target symptomatic and palliative management. We describe the case of a 10-year-old child who presented to our institute with a severe disability of upper limbs due to joint contractures along with several bony masses at various locations of the body but without having any prior complaints of painful soft tissue lesions or the characteristic flare-ups of the disease ever. Identification of typical soft tissue ossified masses in the specific anatomic pattern, along with the presence of short and malformed great toes helped us in reaching the diagnosis. Surgical procedures including biopsies should be strictly avoided in such patients to prevent triggering the development of more lesions, which occurred in our patient after inadvertent removal of the first swelling by an orthopaedic specialist.