Evaluation of the IP-10 mRNA release assay for diagnosis of TB in HIV-infected individuals

HIV-infected individuals are susceptible to Mycobacterium tuberculosis (M.tb) infection and are at high risk of developing active tuberculosis (TB). Interferon-gamma release assays (IGRAs) are auxiliary tools in the diagnosis of TB. However, the performance of IGRAs in HIV-infected individuals is suboptimal, which limits clinical application. Interferon-inducible protein 10 (IP-10) is an alternative biomarker for identifying M.tb infection due to its high expression after stimulation with M.tb antigens. However, whether IP-10 mRNA constitutes a target for the diagnosis of TB in HIV-infected individuals is unknown. Thus, we prospectively enrolled HIV-infected patients with suspected active TB from five hospitals between May 2021 and May 2022, and performed the IGRA test (QFT-GIT) alongside the IP-10 mRNA release assay on peripheral blood. Of the 216 participants, 152 TB patients and 48 non-TB patients with a conclusive diagnosis were included in the final analysis. The number of indeterminate results of IP-10 mRNA release assay (13/200, 6.5%) was significantly lower than that of the QFT-GIT test (42/200, 21.0%) (P = 0.000026). IP-10 mRNA release assay had a sensitivity of 65.3% (95%CI 55.9% – 73.8%) and a specificity of 74.2% (95%CI 55.4% – 88.1%), respectively; while the QFT-GIT test had a sensitivity of 43.2% (95%CI 34.1% – 52.7%) and a specificity of 87.1% (95%CI 70.2% – 96.4%), respectively. The sensitivity of the IP-10 mRNA release assay was significantly higher than that of QFT-GIT test (P = 0.00062), while no significant difference was detected between the specificities of these two tests (P = 0.198). The IP-10 mRNA release assay showed a lower dependence on CD4+ T cells than that of QFT-GIT test. This was evidenced by the fact that the QFT-GIT test had a higher number of indeterminate results and a lower sensitivity when the CD4+ T cells counts were decreased (P < 0.05), while no significant difference in the number of indeterminate results and sensitivity were observed for the IP-10 mRNA release assay among HIV-infected individuals with varied CD4+T cells counts (P > 0.05). Therefore, our study suggested that M.tb specific IP-10 mRNA is a better biomarker for diagnosis of TB in HIV-infected individuals

A consensus protocol for functional connectivity analysis in the rat brain

Task-free functional connectivity in animal models provides an experimental framework to examine connectivity phenomena under controlled conditions and allows for comparisons with data modalities collected under invasive or terminal procedures. Currently, animal acquisitions are performed with varying protocols and analyses that hamper result comparison and integration. Here we introduce StandardRat, a consensus rat functional magnetic resonance imaging acquisition protocol tested across 20 centers. To develop this protocol with optimized acquisition and processing parameters, we initially aggregated 65 functional imaging datasets acquired from rats across 46 centers. We developed a reproducible pipeline for analyzing rat data acquired with diverse protocols and determined experimental and processing parameters associated with the robust detection of functional connectivity across centers. We show that the standardized protocol enhances biologically plausible functional connectivity patterns relative to previous acquisitions. The protocol and processing pipeline described here is openly shared with the neuroimaging community to promote interoperability and cooperation toward tackling the most important challenges in neuroscience

Identification of unique transcriptomic signatures and key genes through RNA sequencing and integrated WGCNA and PPI network analysis in HIV infected lung cancer

Abstract With the widespread use of highly active antiretroviral therapy (HARRT), the survival time of AIDS patients has been greatly extended. However, the incidence of lung cancer in HIV‐infected patients is increasing and has become a major problem threatening the survival of AIDS patients. The aim of this study is to use Weighted Gene Co‐expression Network Analysis (WGCNA) and differential gene analysis to find possible key genes involved in HIV‐infected lung cancer. In this study, using lung tissue samples from five pairs of HIV‐infected lung cancer patients, second‐generation sequencing was performed and transcriptomic data were obtained. A total of 132 HIV‐infected lung cancer‐related genes were screened out by WGCNA and differential gene expression analysis methods. Based on gene annotation analysis, these genes were mainly enriched in mitosis‐related functions and pathways. In addition, in protein–protein interaction (PPI) analysis, a total of 39 hub genes were identified. Among them, five genes (ASPM, CDCA8, CENPF, CEP55, and PLK1) were present in both three hub gene lists (intersection gene, DEGs, and WCGNA module) suggesting that these five genes may become key genes involved in HIV‐infected lung cancer

Recombinant Human Parathyroid Hormone Related Protein 1-34 and 1-84 and Their Roles in Osteoporosis Treatment

<div><p>Osteoporosis is a common disorder characterized by compromised bone strength that predisposes patients to increased fracture risk. Parathyroid hormone related protein (PTHrP) is one of the candidates for clinical osteoporosis treatment. In this study, GST Gene Fusion System was used to express recombinant human PTHrP (hPTHrP) 1-34 and 1-84. To determine whether the recombinant hPTHrP1-34 and 1-84 can enhance renal calcium reabsorption and promote bone formation, we examined effects of recombinant hPTHrP1-34 and 1-84 on osteogenic lineage commitment in a primary bone marrow cell culture system and on osteoporosis treatment. Results revealed that both of recombinant hPTHrP1-34 and 1-84 increased colony formation and osteogenic cell differentiation and mineralization in vitro; however, the effect of recombinant hPTHrP1-84 is a little stronger than that of hPTHrP1-34. Next, ovariectomy was used to construct osteoporosis animal model (OVX) to test activities of these two recombinants in vivo. HPTHrP1-84 administration elevated serum calcium by up-regulating the expression of renal calcium transporters, which resulted in stimulation of osteoblastic bone formation. These factors contributed to augmented bone mass in hPTHrP1-84 treated OVX mice but did not affect bone resorption. There was no obvious bone mass alteration in hPTHrP1-34 treated OVX mice, which may be, at least partly, associated with shorter half-life of hPTHrP1-34 compared to hPTHrP1-84 in vivo. This study implies that recombinant hPTHrP1-84 is more effective than hPTHrP1-34 to enhance renal calcium reabsorption and to stimulate bone formation in vivo.</p></div

Preparation of Recombinant hPTHrP1-34 and 1-84.

<p>(a) Determination of the recombinants by PCR and double-enzyme (EcoR I+ BamH I) digestion. LaneM1, l-Hind III digest DNA marker; Lane1-3, double-enzyme digestion patterns of recombinant <i>hPTHrP1-34</i>, <i>hPTHrP1-84</i> and <i>pGEX-2TK</i>; LaneM2, DL2000 DNA marker; Lane4-5, PCR band patterns of recombinant hPTHrP1-34 and hPTHrP1-84. SDS-PAGE analysis of the GST-hPTHrP1-34 (b) or GST-hPTHrP1-84 (d) fusion protein under inducing condition and stained with coomassie blue. LaneM, molecular weight marker; Lane1, total cell soluble protein of BL21, control; Lanes2–6, cells with <i>pGEX-2TK/hPTHrP1-34</i> or <i>pGEX-2TK/hPTHrP1-84</i> after inducing with IPTG 0 h, 1 h, 2 h, 3 h, 4 h respectively. SDS-PAGE analyses of hPTHrP1-34 (c) or hPTHrP1-84 (e) peptide expression and purification. LaneM, molecular weight marker; Lane1, IPTG induction of BL21 transformed with <i>pGEX-2TK</i> without insert (expression control); Lane2, BL21 transformed with <i>pGEX-2TK/hPTHrP1-34</i> or <i>pGEX-2TK/hPTHrP1-84</i> without IPTG induction (induction control); Lane3, IPTG induction of BL21 transformed with <i>pGEX-2TK/hPTHrP1-34</i> or <i>pGEX-2TK/hPTHrP1-84</i>; Lane4, The proteins not bound to GSTrap FF column; Lane5, Purified hPTHrP1-34 or hPTHrP1-84 obtained after binding to GSTrap FF column and cleaved by thrombin; Lane6, GST-tag eluted from GSTrap FF column. (f) Western blot analysis of purified recombinant hPTHrP1-34 and hPTHrP1-84 with N-terminal hPTHrP antibody.</p

Effects of recombinant hPTHrP1-34 and 1-84 on BMCs.

<p>Cells from 18-day primary BMC cultures incubated in the absence (Control) or presence of 10⁻⁷ M hPTHrP1-34 (hPTHrP1-34) or presence of 10⁻⁷ M hPTHrP1-84 (hPTHrP1-84) on day 0 were stained cytochemically for ALP (a. CFU-f _ALP) and with sirius red for total collagen(c. CFU-f _Col) and with the von Kossa method for calcified colonies(e. CFU-f _Ca) and with methylene blue to show total CFU-f (g. Total CFU-f) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088237#s2" target="_blank">Materials and Methods</a>. The positive number of CFU-f _ALP (b), CFU-f _Col (d), CFU-f _Ca (f) and Total CFU-f number (h) per dish are the mean±SEM of triplicate determinations from three replicate experiments, respectively. (i) Real-time RT-PCR of cells extracts from 14-day primary BMC cultures for the expression of Cbfa I, ALP, Col I and OCN. Messenger RNA expression assessed by real-time RT-PCR is calculated as a ratio to the GAPDH mRNA level and expressed relative to levels of Control group. Each value is the mean ± SEM of three trials. *, P<0.05; **, P<0.01; ***, P<0.001 compared with Control group; #, P<0.05 compared with hPTHrP1-34 group.</p

Effects of recombinant hPTHrP1-34 and 1-84 on osteoclastic bone resorption parameters and half-life analyses of recombinant hPTHrP1-34 and 1-84 in vivo.

<p>(a) Representative micrographs of paraffin embedded sections of vertebrae stained histochemically for TRAP and photographed at a magnification of 200. Scale bars in a represents 50 µm. (b) Number of TRAP positive osteoclasts per mm bone parameter (N.Oc/B.Pm, #/mm) and (c) the surface of osteoclasts relative to the bone surface (Oc.S/B.S, %) were determined in the trabeculae of TRAP-stained vertebrae. Each value is the mean ± SEM of determinations in 6 animals of each group. (d) Real-time RT-PCR was performed on long bone extracts for <i>RANKL</i> and <i>OPG</i> mRNA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088237#s2" target="_blank">Materials and Methods</a>. Messenger RNA expression assessed by real-time RT-PCR analysis is calculated as a ratio to the <i>GAPDH</i> mRNA level and expressed relative to levels of Sham group. Ratio of <i>RANKL/OPG</i> relative mRNA levels was calculated and was presented as the mean ± SEM of determinations in six animals of each group. (e) Plasma immunoreactive PTHrP determined using ELISA after subcutaneous injection of vehicle or hPTHrP1-34 or hPTHrP1-84. Blood samples were drawn at the following time points after injection of study drugs: 15 and 30 minutes, then 1, 2, 4, 6 hours. Each value is the mean ± SEM of determinations in 6 animals of each group. *, P<0.05; **, P<0.01 compared with Sham group.</p

Effects of recombinant hPTHrP1-34 and 1-84 on osteoblastic bone formation parameters.

<p>(a) Representative micrographs of calcein double labeling and (c) sections stained with the von Kossa procedure in the trabeculae were imaged from ethanol fixed and undecalcified LR white resin embedded sections of vertebrae. (b) MAR of trabeculae was determined. (d) Osteoid volume was determined in undecalcified von Kossa-stained sections and is presented as a percent of bone volume (OV/BV, %) of trabeculae. Micrographs of decalcified paraffin sections of vertebrae stained with H&E (e) and histochemically for ALP (g). (f) Number of osteoblasts per mm bone parameter (N.Ob/B.Pm, #/mm) and (h) ALP positive area as a percent of the tissue area were determined in the vertebrae. Scale bars represent 25 µm in a and c and 50 µm in e and g. (i) Real-time RT-PCR of long bone extracts for the expression of <i>Cbfa I</i>, <i>ALP</i>, <i>Col I</i> and <i>OCN</i>. Messenger RNA expression assessed by real-time RT-PCR is calculated as a ratio to the <i>GAPDH</i> mRNA level and expressed relative to levels of Sham group. Each value is the mean ± SEM of determinations in six mice of each group. *, P<0.05; **, P<0.01 compared with Sham group; #, P<0.05; ##, P<0.01 compared with Control group; △, P<0.05 compared with hPTHrP1-34 group.</p

Effects of recombinant hPTHrP1-34 and 1-84 on serum chemistry and on expression of calcium transporters in kidney.

<p>(a) Serum calcium, (b) phosphorus and (c) ALP ratio were determined after 4-week administration in vehicle-treated sham-operated mice (Sham), vehicle-treated OVX mice (Control), hPTHrP1-34-treated OVX mice (hPTHrP1-34) and hPTHrP1-84-treated OVX mice (hPTHrP1-84). (d) Western blots of renal extracts for expression of CB_28K, CB_9K, NCX1 and TRPV5. β-tubulin was used as loading control for Western blots. (e) CB_28K, CB_9K, NCX1 and TRPV5 protein levels relative to β-tubulin protein level and expressed relative to levels of Sham group. Each value is the mean ± SEM of determinations in six mice of each group.*, P<0.05; ***, P<0.001 compared with Sham group; #, P<0.05 compared with Control group; △, p<0.05 compared with hPTHrP1-34 group.</p