Bug in the code: TB blocks DNA repair.

Protecting the cell's genome is crucial for survival, but infection causes damage that compromises genetic integrity. In this issue of Cell Host & Microbe, Lui et al. dissect how Mycobacterium tuberculosis exploits DNA damage using a secreted protein that inhibits DNA repair to create an environment conducive to bacterial replication.

Enhanced immunogenicity of Mycobacterium bovis BCG through CRISPRi mediated depletion of AftC.

Mycobacterium tuberculosis causes the disease tuberculosis and affects a third of the world's population. The recent COVID-19 pandemic exacerbated the situation with a projected 27% increase in tuberculosis related deaths. M. tuberculosis has an elaborate cell wall consisting of peptidoglycan, arabinogalactan and mycolic acids which shield the bacilli from the toxic bactericidal milieu within phagocytes. Amongst, the numerous glycosyltransferase enzymes involved in mycobacterial cell wall biosynthesis, arabinofuranosyltransferase C (aftC) is responsible for the branching of the arabinan domain in both arabinogalactan and lipoarabinomannan. Using Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) we have generated aftC knockdowns in Mycobacterium bovis BCG and demonstrated the generation of a truncated, immunogenic lipoarabinomannan within its cell envelope. The aftC depleted BCG mutants were unable to form characteristic mycobacterial pellicular biofilms and elicit a potent immunostimulatory phenotype compared to wild type M. bovis BCG in a THP1 cell line. This study paves the way to further explore novel BCG mutants as promising vaccine boosters in preventing pulmonary tuberculosis.

Temporal patterns of cortical proliferation of glial cell populations after traumatic brain injury in mice.

TBI (traumatic brain injury) triggers an inflammatory cascade, gliosis and cell proliferation following cell death in the pericontusional area and surrounding the site of injury. In order to better understand the proliferative response following CCI (controlled cortical impact) injury, we systematically analyzed the phenotype of dividing cells at several time points post-lesion. C57BL/6 mice were subjected to mild to moderate CCI over the left sensory motor cortex. At different time points following injury, mice were injected with BrdU (bromodeoxyuridine) four times at 3-h intervals and then killed. The greatest number of proliferating cells in the pericontusional region was detected at 3 dpi (days post-injury). At 1 dpi, NG2+ cells were the most proliferative population, and at 3 and 7 dpi the Iba-1+ microglial cells were proliferating more. A smaller, but significant number of GFAP+ (glial fibrillary acidic protein) astrocytes proliferated at all three time points. Interestingly, at 3 dpi we found a small number of proliferating neuroblasts [DCX+ (doublecortin)] in the injured cortex. To determine the cell fate of proliferative cells, mice were injected four times with BrdU at 3 dpi and killed at 28 dpi. Approximately 70% of proliferative cells observed at 28 dpi were GFAP+ astrocytes. In conclusion, our data suggest that the specific glial cell types respond differentially to injury, suggesting that each cell type responds to a specific pattern of growth factor stimulation at each time point after injury.

Smad proteins differentially regulate transforming growth factor-ß-mediated induction of chondroitin sulfate proteoglycans.

Traumatic injury to the CNS results in increased expression and deposition of chondroitin sulfate proteoglycans (CSPGs) that are inhibitory to axonal regeneration. Transforming growth factor-ß (TGF-ß) has been implicated as a major mediator of these changes, but the mechanisms through which TGF-ß regulates CSPG expression are not known. Using lentiviral expressed Smad-specific ShRNA we show that TGF-ß induction of CSPG expression in astrocytes is Smad-dependent. However, we find a differential dependence of the synthetic machinery on Smad2 and/or Smad3. TGF-ß induction of neurocan and xylosyl transferase 1 required both Smad2 and Smad3, whereas induction of phosphacan and chondroitin synthase 1 required Smad2 but not Smad3. Smad3 knockdown selectively reduced induction of chondroitin-4-sulfotransferase 1 and the amount of 4-sulfated CSPGs secreted by astrocytes. Additionally, Smad3 knockdown in astrocytes was more efficacious in promoting neurite outgrowth of neurons cultured on the TGF-ß-treated astrocytes. Our data implicate TGF-ß Smad3-mediated induction of 4-sulfation as a critical determinant of the permissiveness of astrocyte secreted CSPGs for axonal growth.

Larvicidal activity of Saraca indica, Nyctanthes arbor-tristis, and Clitoria ternatea extracts against three mosquito vector species.

Screening of natural products for mosquito larvicidal activity against three major mosquito vectors Aedes aegypti, Culex quinquefasciatus, and Anopheles stephensi resulted in the identification of three potential plant extracts viz., Saraca indica/asoca, Nyctanthes arbor-tristis, and Clitoria ternatea for mosquito larval control. In the case of S. indica/asoca, the petroleum ether extract of the leaves and the chloroform extract of the bark were effective against the larvae of C. quinquefasciatus with respective LC(50) values 228.9 and 291.5 ppm. The LC(50) values of chloroform extract of N. arbor-tristis leaves were 303.2, 518.2, and 420.2 ppm against A. aegypti, A. stephensi, and C. quinquefasciatus, respectively. The methanol and chloroform extracts of flowers of N. arbor-tristis showed larvicidal activity against larvae of A. stephensi with the respective LC(50) values of 244.4 and 747.7 ppm. Among the methanol extracts of C. ternatea leaves, roots, flowers, and seeds, the seed extract was effective against the larvae of all the three species with LC(50) values 65.2, 154.5, and 54.4 ppm, respectively, for A. stephensi, A. aegypti, and C. quinquefasciatus. Among the three plant species studied for mosquito larvicidal activity, C. ternatea was showing the most promising mosquito larvicidal activity. The phytochemical analysis of the promising methanolic extract of the seed extract was positive for carbohydrates, saponins, terpenoids, tannins, and proteins. In conclusion, bioassay-guided fractionation of effective extracts may result in identification of a useful molecule for the control of mosquito vectors.

Ubiquitination-mediated internalization and degradation of the astroglial glutamate transporter, GLT-1.

Sodium-dependent glutamate uptake is essential for limiting excitotoxicity, and dysregulation of this process has been implicated in a wide array of neurological disorders. The majority of forebrain glutamate uptake is mediated by the astroglial glutamate transporter, GLT-1. We and others have shown that this transporter undergoes endocytosis and degradation in response to activation of protein kinase C (PKC), however, the mechanisms involved remain unclear. In the current study, transfected C6 glioma cells or primary cortical cultures were used to show that PKC activation results in incorporation of ubiquitin into GLT-1 immunoprecipitates. Mutation of all 11 lysine residues in the amino and carboxyl-terminal domains to arginine (11R) abolished this signal. Selective mutation of the seven lysine residues in the carboxyl terminus (C7K-R) did not eliminate ubiquitination, but it completely blocked PKC-dependent internalization and degradation. Two families of variants of GLT-1 were prepared with various lysine residues mutated to arginine. Analyses of these constructs indicated that redundant lysine residues in the carboxyl terminus were sufficient for the appearance of ubiquitinated product and degradation of GLT-1. Together these data define a novel mechanism by which the predominant forebrain glutamate transporter can be rapidly targeted for degradation.

Internalization and degradation of the glutamate transporter GLT-1 in response to phorbol ester.

Activation of protein kinase C (PKC) decreases the activity and cell surface expression of the predominant forebrain glutamate transporter, GLT-1. In the present study, C6 glioma were used as a model system to define the mechanisms that contribute to this decrease in cell surface expression and to determine the fate of internalized transporter. As was previously observed, phorbol 12-myristate 13-acetate (PMA) caused a decrease in biotinylated GLT-1. This effect was blocked by sucrose or by co-expression with a dominant-negative variant of dynamin 1, and it was attenuated by co-expression with a dominant-negative variant of the clathrin heavy chain. Depletion of cholesterol with methyl-beta-cyclodextrin, co-expression with a dominant-negative caveolin-1 mutant (Cav1/S80E), co-expression with dominant-negative variants of Eps15 (epidermal-growth-factor receptor pathway substrate clone 15), or co-expression with dominant-negative Arf6 (T27N) had no effect on the PMA-induced loss of biotinylated GLT-1. Long-term treatment with PMA caused a time-dependent loss of biotinylated GLT-1 and decreased the levels of GLT-1 protein. Inhibitors of lysosomal degradation (chloroquine or ammonium chloride) or co-expression with a dominant-negative variant of a small GTPase implicated in trafficking to lysosomes (Rab7) prevented the PMA-induced decrease in protein and caused an intracellular accumulation of GLT-1. These results suggest that the PKC-induced redistribution of GLT-1 is dependent upon clathrin-mediated endocytosis. These studies identify a novel mechanism by which the levels of GLT-1 could be rapidly down-regulated via lysosomal degradation. The possibility that this mechanism may contribute to the loss of GLT-1 observed after acute insults to the CNS is discussed.

Constitutive endocytosis and recycling of the neuronal glutamate transporter, excitatory amino acid carrier 1.

The neuronal glutamate transporter, excitatory amino acid carrier 1 (EAAC1), has a diverse array of physiologic and metabolic functions. There is evidence that there is a relatively large intracellular pool of EAAC1 both in vivo and in vitro, that EAAC1 cycles on and off the plasma membrane, and that EAAC1 cell surface expression can be rapidly regulated by intracellular signals. Despite the possible relevance of EAAC1 trafficking to both physiologic and pathologic processes, the cellular machinery involved has not been defined. In the present study, we found that agents that disrupt clathrin-dependent endocytosis or plasma membrane cholesterol increased steady-state levels of biotinylated EAAC1 in C6 glioma cells and primary neuronal cultures. Acute depletion of cholesterol increased the V(max) for EAAC1-mediated activity and had no effect on Na(+)-dependent glycine transport in the same system. These agents also impaired endocytosis as measured using a reversible biotinylating reagent. Co-expression with dominant-negative variants of dynamin or the clathrin adaptor, epidermal growth factor receptor pathway substrate clone 15, increased the steady-state levels of biotinylated myc-EAAC1. EAAC1 immunoreactivity was found in a subcellular fraction enriched in early endosome antigen 1 (EEA1) isolated by differential centrifugation and partially co-localized with EEA1. Co-expression of a dominant-negative variant of Rab11 (Rab11 S25N) reduced steady-state levels of biotinylated myc-EAAC1 and slowed constitutive delivery of myc-EAAC1 to the plasma membrane. Together, these observations suggest that EAAC1 is constitutively internalized via a clathrin- and dynamin-dependent pathway into early endosomes and that EAAC1 is trafficked back to the cell surface via the endocytic recycling compartment in a Rab11-dependent mechanism. As one defines the machinery required for constitutive trafficking of EAAC1, it may be possible to determine how intracellular signals regulate EAAC1 cell surface expression.

Evidence that protein kinase Calpha interacts with and regulates the glial glutamate transporter GLT-1.

Many of the sodium-dependent neurotransmitter transporters are rapidly (within minutes) regulated by protein kinase C (PKC), with changes in activity being correlated with changes in transporter trafficking to or from the plasma membrane. Our recent studies suggest that one of the classical subtypes of PKC, PKCalpha, may selectively mediate redistribution of the neuronal glutamate transporter, excitatory amino acid carrier (EAAC)1, and show that PKCalpha can be co-immunoprecipitated with EAAC1. When the glial glutamate transporter GLT-1a is transfected into C6 glioma cells, this transporter is internalized in response to activation of PKC, but the PKC subtype involved in this regulation is unknown. In the present study, expression of the phorbol ester-activated subtypes of PKC was examined in C6 glioma transfected with GLT-1. Of the classical subtypes, only PKCalpha was detected, and of the non-classical subtypes, PKCdelta and PKCepsilon were detected. In this system, phorbol ester-dependent internalization of GLT-1 was blocked by a general inhibitor of PKCs (bisindolylmaleimide II) and by concentrations of Gö6976 that selectively block classical PKCs, but not by an inhibitor of PKCdelta (rottlerin). PKCalpha immunoreactivity was found in GLT-1 immunoprecipitates obtained from transfected C6 cells and from crude rat brain synaptosomes, a milieu that better mimics in vivo conditions. The amount of PKCalpha in both types of immunoprecipitate was modestly increased by phorbol ester, and this increase was blocked by a PKC antagonist. These studies suggest that PKCalpha may be required for the regulated redistribution of GLT-1.

Differential regulation of GLAST immunoreactivity and activity by protein kinase C: evidence for modification of amino and carboxyl termini.

Many neurotransmitter transporters, including the GLT-1 and EAAC1 subtypes of the glutamate transporter, are regulated by protein kinase C (PKC) and these effects are associated with changes in cell surface expression. In the present study, the effects of PKC activation on the glutamate aspartate transporter (GLAST) subtype of glutamate transporter were examined in primary astrocyte cultures. Acute (30 min) exposure to the phorbol 12-myristate 13-acetate (PMA) increased (approximately 20%) transport activity but had the opposite effect on both total and cell surface immunoreactivity. Chronic treatment (6 or 24 h) with PMA had no effect on transport activity but caused an even larger decrease in total and cell surface immunoreactivity. This loss of immunoreactivity was observed using antibodies directed against three different cytoplasmic epitopes, and was blocked by the PKC antagonist, bisindolylmaleimide II. We provide biochemical and pharmacological evidence that the activity observed after treatment with PMA is mediated by GLAST. Two different flag-tagged variants of the human homolog of GLAST were introduced into astrocytes using lentiviral vectors. Although treatment with PMA caused a loss of transporter immunoreactivity, flag immunoreactivity did not change in amount or size. Together, these studies suggest that activation of PKC acutely up-regulates GLAST activity, but also results in modification of several different intracellular epitopes so that they are no longer recognized by anti-GLAST antibodies. We found that exposure of primary cultures of neurons/astrocytes to transient hypoxia/glucose deprivation also caused a loss of GLAST immunoreactivity that was attenuated by the PKC antagonist, bisindolylmaleimide II, suggesting that some acute insults previously thought to cause a loss of GLAST protein may mimic the phenomenon observed in the present study.

Rottlerin, an inhibitor of protein kinase Cdelta (PKCdelta), inhibits astrocytic glutamate transport activity and reduces GLAST immunoreactivity by a mechanism that appears to be PKCdelta-independent.

Protein kinase C (PKC) regulates the activity and/or cell surface expression of several different neurotransmitter transporters, including subtypes of glutamate transporters. In the present study, the effects of pharmacological inhibitors of PKC were studied in primary astrocyte cultures that express the glutamate aspartate transporter (GLAST) subtype of glutamate transporter. We found that general inhibitors of PKC, bisindolylmaleimide I (Bis I), bisindolylmaleimide II (Bis II), staurosporine and an inhibitor of classical PKCs, Gö6976, had no effect on Na+-dependent glutamate transport activity. However, rottlerin, a putative specific inhibitor of PKCdelta, decreased transport activity with an IC50 value (less than 10 micro m) that is comparable to that reported for inhibition of PKCdelta. The effect of rottlerin was very rapid (maximal effect within 5 min) and was due to a decrease in the capacity (Vmax) for transport. Rottlerin also caused a drastic loss of GLAST immunoreactivity within 5 min, suggesting that rottlerin accelerates GLAST degradation/proteolysis. Rottlerin had no effect on cell surface or total expression of the transferrin receptor, providing evidence that the effect on GLAST cannot be attributed to a non-specific internalization/degradation of plasma membrane proteins. Down-regulation of PKCdelta with chronic phorbol ester treatment did not block rottlerin-mediated inhibition of transport activity. These results suggest a novel mechanism for regulation of the GLAST subtype of glutamate transporter and indicate that there is a rottlerin target that is capable of controlling the levels of GLAST by controlling the rate of degradation or limited proteolysis. It appears that the target for rottlerin may not be PKCdelta.

Vitamin D deficiency reduces insulin secretion and turnover in rats.

Vitamin D deficiency can result in low serum insulin levels, which lead to impaired glucose tolerance. The present study was conducted to assess the mechanisms of this impairment. Pancreatic secretion of insulin and insulin turnover in vitamin D-deficient rats were studied. Insulin secretion was found to be significantly decreased in vitamin D-deficient rats compared to control rats. Supplementation with calcium alone could reverse the defect. The impaired insulin secretion observed in vitamin D deficiency was not specific to glucose stimulation but was also seen in response to arginine. Insulin turnover was studied in vivo using A14-(125)I-insulin. The pharmacokinetic parameters (elimination rate constant K, insulin biological half-life, t1/2, plasma concentrations of insulin at time zero I0) were calculated from the plasma radioactivity. The vitamin D-deficient rats had a lower degradation rate than controls. These findings suggest that vitamin D deficiency leads to reduction in both insulin secretion and turnover, and that the reduced turnover could be an adaptive mechanism to conserve the low amounts of insulin secreted.

Diverse effects of mild and potent goitrogens on blood-brain barrier nutrient transport.

Populations living in goitre endemic areas consume foods rich in a variety of goitrogens of different potencies and some are severely hypothyroid. Recently we observed in Wistar/NIN rats that chronic feeding of KSCN to dams produced only a moderate hypothyroidism and decreased the transport of 2-deoxy-D-glucose (2-DG) across the blood-brain barrier (BBB) in the offspring. The present studies were conducted to assess whether severe hypothyroidism would have greater effect on BBB nutrient transport. It has now been observed that weaning the pups of KSCN fed dams on to KSCN diet for four weeks had no further effect either on their thyroid status or the BBB 2-DG transport. However, feeding KSCN to rats through two generations produced somewhat severe hypothyroidism in F2 pups than that in F1 pups. Interestingly, unlike in F1 pups, the BBB transport of all the three nutrients tested (2-DG, Leu and Tyr) was significantly decreased in F2 pups, albeit to a small extent (10-15%). On the other hand the potent goitrogen: methyl mercaptoimidazole (MMI) even on short term feeding to pregnant dams produced very severe hypothyroidism in the offspring [Serum T4:0.55+/-0.09 microg/dl vs 4.96+/-0.85 in controls]. Surprisingly, the BBB transport of 2-DG, Leu, Tyr and also sucrose, the background marker, was significantly increased in these pups (20-30%). The diverse effects of goitrogen-induced moderate and severe hypothyroidism observed here on the BBB nutrient transport probably suggest different mechanisms for iodine deficiency disorders of different aetiologies and hence the need for discrete approaches for their management.

Dietary goitrogen-induced changes in the transport of 2-deoxy-D-glucose and amino acids across the rat blood-brain barrier.

The hypothesis that a defect in the rate-limiting blood-brain barrier (BBB) nutrient transport may be one of the factors responsible for the brain defects seen in some iodine deficiency disorders was tested in Wistar/NIN rats fed potassium thiocyanate (KSCN), a synthetic goitrogen. The BBB nutrient transport was measured by the brain uptake index (BUI) method. Feeding KSCN to female rats (from weaning) through their growth, pregnancy and lactation (G1) but not from conception (G2) or parturition (G3) resulted in a significant decrease (approximately 23%) in the BBB transport of 2-deoxy-D-glucose (2-DG) in their offspring at weaning, compared with controls (C). Post-weaning KSCN-feeding (G4) of control pups did not affect their BBB 2-DG transport (BUI: 36.2 +/- 4.98, vs 38.8 +/- 4.11). The effects of different KSCN regimens on BBB transport of leucine (leu), tyrosine (tyr) and sucrose (background marker) were inconsistent, of smaller magnitude (approximately 10%) and appeared to be of little significance. Withdrawing KSCN from the diet of chronically KSCN-fed (G1) mothers from conception (G5) or parturition (G6) prevented the impairment of BBB 2-DG transport in pups (BUI: 27.0 +/- 4.98, 20.8 +/- 3.27, 26.9 +/- 3.99 and 28.3 +/- 3.47 in C, G1, G5 and G6 pups, respectively); this was reversed by feeding the control diet to G1 pups from weaning. Withdrawal of dietary KSCN did not affect BBB transport of leu, tyr and sucrose. The decreased BBB transport of 2-DG in G1 pups appears to be due to a decrease in affinity (Kt app 5.46 vs 4.15 mM) rather than in the capacity (Tmax app 0.94 vs 0.91 mumoles/g/min) of the transport system. Intracarotid injections of KSCN per se had no effect on BBB 2-DG transport, suggesting that the effects may be secondary to the altered thyroid status of the animal.

[Captopril treatment--2-year period of observation]. / Leczenie kaptoprylem--2-letni okres obserwacji.

Captopril was administered to 50 carefully selected patients with severe circulatory failure (18 patients classified as class III and 32 as class IV according to NYHA) in daily dose of 37.5-75 mg for two years. Patients were also given digoxin, diuretic agents and iso-dinitrosorbide. Clinical improvement increased with duration of captopril therapy. A significant improvement following the correction of therapy was achieved in 15% of patients, following one month in 28%, three months--in 70%, and after 1 and 2 years in 84% of the treated patients. All patients survived for one year, and 44--for two years (88%). Clinical improvement was manifested by: diminished of dyspnoea, edema, pulmonary and liver congestion, increase in left ventricle ejection fraction, change of disease staging by one or two NYHA classes, and reduced ventricular rate during atrial fibrillation (in 30% of patients within one year). More noticeable improvement was seen in patients with baseline ejection fraction > 40% than those with EF < 30%, in hypertensive patients than normotensive, and in patients classified to III NYHA class. Ejection fraction increased from 37.9 +/- 9.2% before the treatment to 54.6 +/- 7.7% after a two-year captopril therapy (p < .01). Captopril greatly contributes to the successful therapy of the chronic severe heart failure.