An Inverse Agonist Ligand of the PTH Receptor Partially Rescues Skeletal Defects in a Mouse Model of Jansen’s Metaphyseal Chondrodysplasia

Jansen’s metaphyseal chondrodysplasia (JMC) is a rare disease of bone and mineral ion physiology that is caused by activating mutations in PTHR1. Ligand‐independent signaling by the mutant receptors in cells of bone and kidney results in abnormal skeletal growth, excessive bone turnover, and chronic hypercalcemia and hyperphosphaturia. Clinical features further include short stature, limb deformities, nephrocalcinosis, and progressive losses in kidney function. There is no effective treatment option available for JMC. In previous cell‐based assays, we found that certain N‐terminally truncated PTH and PTHrP antagonist peptides function as inverse agonists and thus can reduce the high rates of basal cAMP signaling exhibited by the mutant PTHR1s of JMC in vitro. Here we explored whether one such inverse agonist ligand, [Leu11,dTrp12,Trp23,Tyr36]‐PTHrP(7‐36)NH2 (IA), can be effective in vivo and thus ameliorate the skeletal abnormalities that occur in transgenic mice expressing the PTHR1‐H223R allele of JMC in osteoblastic cells via the collagen‐1α1 promoter (C1HR mice). We observed that after 2 weeks of twice‐daily injection and relative to vehicle controls, the IA analog resulted in significant improvements in key skeletal parameters that characterize the C1HR mice, because it reduced the excess trabecular bone mass, bone marrow fibrosis, and levels of bone turnover markers in blood and urine. The overall findings provide proof‐of‐concept support for the notion that inverse agonist ligands targeted to the mutant PTHR1 variants of JMC can have efficacy in vivo. Further studies of such PTHR1 ligand analogs could help open paths toward the first treatment option for this debilitating skeletal disorder. © 2019 American Society for Bone and Mineral Research.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154322/1/jbmr3913-sup-0001-Supinfo.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154322/2/jbmr3913.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154322/3/jbmr3913_am.pd

Molecular epidemiology, drug susceptibility and economic aspects of tuberculosis in mubende district, Uganda

<div><p>Background</p><p>Tuberculosis (TB) remains a global public health problem whose effects have major impact in developing countries like Uganda. This study aimed at investigating genotypic characteristics and drug resistance profiles of <i>Mycobacterium tuberculosis</i> isolated from suspected TB patients. Furthermore, risk factors and economic burdens that could affect the current control strategies were studied.</p><p>Methods</p><p>TB suspected patients were examined in a cross-sectional study at the Mubende regional referral hospital between February and July 2011. A questionnaire was administered to each patient to obtain information associated with TB prevalence. Isolates of <i>M. tuberculosis</i> recovered during sampling were examined for drug resistance to first line anti-TB drugs using the BACTEC-MGIT960^TMsystem. All isolates were further characterized using deletion analysis, spoligotyping and MIRU-VNTR analysis. Data were analyzed using different software; MIRU-VNTR <i>plus</i>, SITVITWEB, BioNumerics and multivariable regression models.</p><p>Results</p><p><i>M. tuberculosis</i> was isolated from 74 out of 344 patients, 48 of these were co-infected with HIV. Results from the questionnaire showed that previously treated TB, co-infection with HIV, cigarette smoking, and overcrowding were risk factors associated with TB, while high medical related transport bills were identified as an economic burden. Out of the 67 isolates that gave interpretable results, 23 different spoligopatterns were detected, nine of which were novel patterns. T2 with the sub types Uganda-I and Uganda-II was the most predominant lineage detected. Antibiotic resistance was detected in 19% and multidrug resistance was detected in 3% of the isolates.</p><p>Conclusion</p><p>The study detected <i>M. tuberculosis</i> from 21% of examined TB patients, 62% of whom were also HIV positive. There is a heterogeneous pool of genotypes that circulate in this area, with the T2 lineage being the most predominant. High medical related transport bills and drug resistance could undermine the usefulness of the current TB strategic interventions.</p></div

Tissue Harvester with Functional Valve (THFV): Shidham's device for reproducibly higher specimen yield by fine needle aspiration biopsy with easy to perform steps

BACKGROUND: Fine needle aspiration biopsy (FNAB) cytology has been a highly effective methodology for tissue diagnosis and for various ancillary studies including molecular tests. In addition to other benefits, FNAB predominantly retrieves the diagnostic loosely cohesive cells in the lesion as compared to the adjacent supporting stroma with relatively higher cohesiveness. However, FNAB procedure performed with currently available resources is highly skill dependent with inter-performer variability, which compromises its full potential as a diagnostic tool. In this study we report a device overcoming these limitations. METHODS: 'Tissue Harvester with Functional Valve' (THFV) was evaluated as part of a phase 1 National Institute of Health (NIH) research grant under Small Business Technology Transfer (STTR) Program. Working prototypes of the device were prepared. Each of the four cytopathologists with previous cytopathology fellowship training and experience in performing FNAB evaluated 5 THFV and 5 hypodermic needles resulting in 40 specimens (20 with THFV, 20 with hypodermic needles). A piece of fresh cattle liver stuffed in latex glove was used as the specimen. Based on these results a finished design was finalized. RESULTS: The smears and cell blocks prepared from the specimens obtained by THFV were superior in terms of cellularity to specimens obtained with hypodermic needles. The tissuecrit of specimens obtained with THFV ranged from 70 to 100 μl (mean 87, SD 10), compared to 17 to 30 μl (mean 24, SD 4) with conventional hypodermic needles (p < .0001, Student t-test). The technical ease [on a scale of 1 (easy) to 5 (difficult)] with THFV ranged from 1 to 2 as compared to 2 to 3 with hypodermic needles. CONCLUSION: The specimen yield with the new THFV was significantly higher when compared to hypodermic needles. Also, the FNAB procedure with THFV was relatively easier in comparison with hypodermic needles. The final version of Shidham's THFV device would improve the FNAB specimen yield by eliminating the skill factor. The increased specimen yield by this device would also facilitate wider application of FNAB specimens for various ancillary tests, including molecular tests

Thioflavine-T and Congo Red reveal the polymorphism of insulin amyloid fibrils when probed by polarization-resolved fluorescence microscopy.

International audienceAmyloid fibrils are protein misfolding structures that involve a β-sheet structure and are associated with the pathologies of various neurodegenerative diseases. Here we show that Thioflavine-T and Congo Red, two major dyes used to image fibrils by fluorescence assays, can provide deep structural information when probed by means of polarization-resolved fluorescence microscopy. Unlike fluorescence anisotropy or fluorescence detected linear dichroism imaging, this technique allows to retrieve simultaneously both mean orientation and orientation dispersion of the dye, used here as a reporter of the fibril structure. We have observed that insulin amyloid fibrils exhibit a homogeneous behavior over the fibrils' length, confirming their structural uniformity. In addition, these results reveal the existence of various structures among the observed fibrils' population, in spite of a similar aspect when imaged with conventional fluorescence microscopy. This optical nondestructive technique opens perspectives for in vivo structural analyses or high throughput screening

Identification of an Amphipathic Helix Important for the Formation of Ectopic Septin Spirals and Axial Budding in Yeast Axial Landmark Protein Bud3p

Correct positioning of polarity axis in response to internal or external cues is central to cellular morphogenesis and cell fate determination. In the budding yeast Saccharomyces cerevisiae, Bud3p plays a key role in the axial bud-site selection (axial budding) process in which cells assemble the new bud next to the preceding cell division site. Bud3p is thought to act as a component of a spatial landmark. However, it is not clear how Bud3p interacts with other components of the landmark, such as the septins, to control axial budding. Here, we report that overexpression of Bud3p causes the formation of small septin rings (∼1 µm in diameter) and arcs aside from previously reported spiral-like septin structures. Bud3p closely associates with the septins in vivo as Bud3p colocalizes with these aberrant septin structures and forms a complex with two septins, Cdc10p and Cdc11p. The interaction of Bud3p with the septins may involve multiple regions of Bud3p including 1–858, 850–1220, and 1221–1636 a.a. since they all target to the bud neck but exhibit different effects on septin organization when overexpressed. In addition, our study reveals that the axial budding function of Bud3p is mediated by the N-terminal region 1–858. This region shares an amphipathic helix (850–858) crucial for bud neck targeting with the middle portion 850–1103 involved in the formation of ectopic septin spirals and rings. Interestingly, the Dbl-homology domain located in 1–858 is dispensable for axial bud-site selection. Our findings suggest that multiple regions of Bud3p ensure efficient targeting of Bud3p to the bud neck in the assembly of the axial landmark and distinct domains of Bud3p are involved in axial bud-site selection and other cellular processes

A Molecular Epidemiological and Genetic Diversity Study of Tuberculosis in Ibadan, Nnewi and Abuja, Nigeria

Background Nigeria has the tenth highest burden of tuberculosis (TB) among the 22 TB high-burden countries in the world. This study describes the biodiversity and epidemiology of drug-susceptible and drug-resistant TB in Ibadan, Nnewi and Abuja, using 409 DNAs extracted from culture positive TB isolates. Methodology/Principal Findings DNAs extracted from clinical isolates of Mycobacterium tuberculosis complex were studied by spoligotyping and 24 VNTR typing. The Cameroon clade (CAM) was predominant followed by the M. africanum (West African 1) and T (mainly T2) clades. By using a smooth definition of clusters, 32 likely epi-linked clusters related to the Cameroon genotype family and 15 likely epi-linked clusters related to other “modern” genotypes were detected. Eight clusters concerned M. africanum West African 1. The recent transmission rate of TB was 38%. This large study shows that the recent transmission of TB in Nigeria is high, without major regional differences, with MDR-TB clusters. Improvement in the TB control programme is imperative to address the TB control problem in Nigeria

Mycobacterium tuberculosis causing tuberculous lymphadenitis in Maputo, Mozambique

BACKGROUND: The zoonosis bovine tuberculosis (TB) is known to be responsible for a considerable proportion of extrapulmonary TB. In Mozambique, bovine TB is a recognised problem in cattle, but little has been done to evaluate how Mycobacterium bovis has contributed to human TB. We here explore the public health risk for bovine TB in Maputo, by characterizing the isolates from tuberculous lymphadenitis (TBLN) cases, a common manifestation of bovine TB in humans, in the Pathology Service of Maputo Central Hospital, in Mozambique, during one year. RESULTS: Among 110 patients suspected of having TBLN, 49 had a positive culture result. Of those, 48 (98 %) were positive for Mycobacterium tuberculosis complex and one for nontuberculous mycobacteria. Of the 45 isolates analysed by spoligotyping and Mycobacterial Interspersed Repetitive Unit - Variable Number Tandem Repeat (MIRU-VNTR), all were M. tuberculosis. No M. bovis was found. Cervical TBLN, corresponding to 39 (86.7 %) cases, was the main cause of TBLN and 66.7 % of those where from HIV positive patients. We found that TBLN in Maputo was caused by a variety of M. tuberculosis strains. The most prevalent lineage was the EAI (n?=?19; 43.2 %). Particular common spoligotypes were SIT 48 (EAI1_SOM sublineage), SIT 42 (LAM 9), SIT 1 (Beijing) and SIT53 (T1), similar to findings among pulmonary cases. CONCLUSIONS: M. tuberculosis was the main etiological agent of TBLN in Maputo. M. tuberculosis genotypes were similar to the ones causing pulmonary TB, suggesting that in Maputo, cases of TBLN arise from the same source as pulmonary TB, rather than from an external zoonotic source. Further research is needed on other forms of extrapulmonary TB and in rural areas where there is high prevalence of bovine TB in cattle, to evaluate the risk of transmission of M. bovis from cattle to humans.Swedish International Development Cooperation Agency / Department for Research Cooperation (Sida/SAREC) through Eduardo Mondlane University and Karolinska Institutet Research and Training (KIRT) collaboratio

Indifferentiability of Iterated Even-Mansour Ciphers with Non-Idealized Key-Schedules: Five Rounds are Necessary and Sufficient

We prove that the 5-round iterated Even-Mansour (IEM) construction (which captures the high-level structure of the class of key-alternating ciphers) with a non-idealized key-schedule (such as the trivial key-schedule, where all round keys are equal) is indifferentiable from an ideal cipher. In a separate result, we also prove that five rounds are necessary by describing an attack against the corresponding 4-round construction. This closes the gap regarding the exact number of rounds for which the IEM construction with a non-idealized key-schedule is indifferentiable from an ideal cipher, which was previously only known to lie between four and twelve

Genomic Diversity of Mycobacterium tuberculosis Complex Strains in Cantabria (Spain), a Moderate TB Incidence Setting

Background Tuberculosis (TB) control strategies are focused mainly on prevention, early diagnosis, compliance to treatment and contact tracing. The objectives of this study were to explore the frequency and risk factors of recent transmission of clinical isolates of Mycobacterium tuberculosis complex (MTBC) in Cantabria in Northern Spain from 2012 through 2013 and to analyze their clonal complexity for better understanding of the transmission dynamics in a moderate TB incidence setting. Methods DNA from 85 out of 87 isolates from bacteriologically confirmed cases of MTBC infection were extracted directly from frozen stocks and genotyped using the mycobacterial interspersed repetitive units-variable number tandem repeat (MIRU-VNTR) method. The MIRUVNTRplus database tool was used to identify clusters and lineages and to build a neighbor joining (NJ) phylogenetic tree. In addition, data were compared to the SITVIT2 database at the Pasteur Institute of Guadeloupe. Results The rate of recent transmission was calculated to 24%. Clustering was associated with being Spanish-born. A high prevalence of isolates of the Euro-American lineage was found. In addition, MIRU-VNTR profiles of the studied isolates corresponded to previously found MIRU-VNTR types in other countries, including Spain, Belgium, Great Britain, USA, Croatia, South Africa and The Netherlands. Six of the strains analyzed represented clonal variants. Conclusion Transmission of MTBC is well controlled in Cantabria. The majority of TB patients were born in Spain. The population structure of MTBC in Cantabria has a low diversity of major clonal lineages with the Euro-American lineage predominating