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Runx1 is highly expressed in osteoblasts, however, its function in osteogenesis is unclear. We generated mesenchymal progenitor-specific ( Runx1 f/f Twist2-Cre) and osteoblast-specific ( Runx1 f/f Col1α1-Cre ) conditional knockout (Runx1 CKO) mice. The mutant CKO mice with normal skeletal development displayed a severe osteoporosis phenotype at postnatal and adult stages. Runx1 CKO resulted in decreased osteogenesis and increased adipogenesis. RNA-sequencing analysis, Western blot, and qPCR validation of Runx1 CKO samples showed that Runx1 regulates BMP signaling pathway and Wnt/β-catenin signaling pathway. ChIP assay revealed direct binding of Runx1 to the promoter regions of Bmp7 , Alk3 , and Atf4 , and promoter mapping demonstrated that Runx1 upregulates their promoter activity through the binding regions. Bmp7 overexpression rescued Alk3, Runx2, and Atf4 expression in Runx1 -deficient BMSCs. Runx2 expression was decreased while Runx1 was not changed in Alk3 deficient osteoblasts. Atf4 overexpression in Runx1 -deficient BMSCs did not rescue expression of Runx1, Bmp7, and Alk3. Smad1/5/8 activity was vitally reduced in Runx1 CKO cells, indicating Runx1 positively regulates the Bmp7/Alk3/Smad1/5/8/Runx2/ATF4 signaling pathway. Notably, Runx1 overexpression in Runx2 -/- osteoblasts rescued expression of Atf4, OCN, and ALP to compensate Runx2 function. Runx1 CKO mice at various osteoblast differentiation stages reduced Wnt signaling and caused high expression of C/ebpα and Pparγ and largely increased adipogenesis. Co-culture of Runx1 -deficient and wild-type cells demonstrated that Runx1 regulates osteoblast−adipocyte lineage commitment both cell-autonomously and non-autonomously. Notably, Runx1 overexpression rescued bone loss in OVX-induced osteoporosis. This study focused on the role of Runx1 in different cell populations with regards to BMP and Wnt signaling pathways and in the interacting network underlying bone homeostasis as well as adipogenesis, and has provided new insight and advancement of knowledge in skeletal development. Collectively, Runx1 maintains adult bone homeostasis from bone loss though up-regulating Bmp7/Alk3/Smad1/5/8/Runx2/ATF4 and WNT/β-Catenin signaling pathways, and targeting Runx1 potentially leads to novel therapeutics for osteoporosis.

Purulent pericarditis is an extremely rare entity with only a few reported cases so far. This condition deserves prompt diagnosis because of its significant mortality rate if left untreated. A 76-year-old man with a past medical history of coronary artery disease (CAD) with percutaneous coronary intervention (PCI) to the left anterior descending artery (LAD) and right circumflex artery (RCA), ischemic cardiomyopathy with moderately reduced ejection fraction (EF 45-50%), peripheral artery disease (PAD), COVID-19 pneumonia complicated by fibrotic lung disease (on 3 liters of home oxygen), type-2 diabetes mellitus (T2DM), hypertension (HTN), hyperlipidemia (HLD), and chronic kidney disease (CKD) stage III presented with complaints of pleuritic chest pain and shortness of breath. On hospital day 1, he was afebrile and hemodynamically stable with physical exam remarkable for bibasilar crackles and dry gangrene of his right first toe. He developed progressive altered mental status, hypotension, oliguric renal failure, and respiratory distress on hospital day 6. On exam at this time, he had an elevated jugular venous distension (JVD) of 12-14 cm water, pericardial friction rub with decreased heart sounds, and orthopnea; all were consistent with cardiac tamponade clinically. An electrocardiogram (EKG) showed new ST elevations in leads I, II, and aVL with ST depression in aVR and V1 with only mild elevation in troponin I to 0.07 ng/mL. A transthoracic echocardiogram (TTE) was done on hospital day 7 and showed a moderate sized pericardial effusion with inferior vena cava (IVC) enlargement but no atrial collapse, ventricular collapse, IVC collapse, or respiratory variation in the mitral and tricuspid inflow velocities. Blood cultures grew methicillin-resistant Staphylococcus aureus (MRSA) on hospital day 6, and he was started on intravenous (IV) vancomycin. The differential diagnosis for his enlarging pericardial effusion included purulent pericarditis, uremic pericarditis, or hemorrhagic effusion. He had urgent diagnostic and therapeutic pericardiocentesis with removal of 350 milliliters of fluid. The pericardial fluid was cloudy, tan-brown with a gram stain showing gram-positive cocci in clusters and cultures growing MRSA, which confirmed the diagnosis of purulent pericarditis secondary to MRSA infection. After the pericardiocentesis, his blood pressure, respiratory distress, and renal failure improved. The source of the bacteremia was from osteomyelitis of his gangrenous, right toe with bone biopsy growing both MRSA and Streptococcus anginosus. He underwent toe amputation for definitive source control. He was discharged on hospital day 24 with a plan to complete 6 weeks of IV vancomycin.

Cathepsins play a role in regulation of cell function through their presence in the cell nucleus. However, the role of Cathepsin K (Ctsk) as an epigenetic regulator in osteoclasts remains unknown. Our data demonstrated that mice have a striking phenotype with a 5-fold increase in bone volume compared with WT. RNA-seq analysis of , and osteoclasts revealed their distinct functions in gene expression regulation, including reduced expression, increased expression, and in signaling pathways activity regulation. Western blots and qPCR data validated these changes. ATAC-seq profiling of , and osteoclasts indicated the changes resulted from reduced chromatin openness in the promoter region of and increased chromatin openness in Nfatc1 promoter in osteoclasts compared to that in osteoclasts of WT, and . We found co-localization of Ctsk with c-Fos and cleavage of H3K27me3 in wild-type osteoclasts. Remarkably, cleavage of H3K27me3 was blocked in osteoclasts of and mice, suggesting that Ctsk may epigenetically regulate distinctive groups of genes' expression by regulating proteolysis of H3K27me3. double knockout dramatically protects against ovariectomy induced bone loss. We found that Ctsk may function as an essential epigenetic regulator in modulating levels of H3K27me3 in osteoclast activation and maintaining bone homeostasis. Our study revealed complementary and unique functions of Ctsk as epigenetic regulators for maintaining osteoclast activation and bone homeostasis by orchestrating multiple signaling pathways and targeting both Ctsk and Mmp9 is a novel therapeutic approach for osteolytic diseases such as osteoporosis.

[This corrects the article DOI: 10.1371/journal.pgen.1009233.].[This corrects the article DOI: 10.1371/journal.pgen.1009233.].

Prevention of hepatitis B virus (HBV) reinfection is important for long-term outcomes following liver transplantation (LT). Hepatitis B immunoglobulin (HBIG) is used among recipients who have (i) native HBV disease, (ii) hepatitis B core antibody positivity (HBcAb positivity), or (iii) received HBcAb positive organs. Nucleos(t)ide analogue (NA) monotherapy is emerging for treating patients in this setting. There is no generalized consensus on the ideal dosage of HBIG. The aim of this study was to evaluate the efficacy of low-dose HBIG (1560 international unit [IU]) for post-LT HBV prevention. HBcAb positive patients who received either HBcAb positive or hepatitis B core antibody negative (HBcAb negative) organs and HBcAb negative patients who received HBcAb positive organs between January 2016 and December 2020 were reviewed. Pre-LT HBV serologies were collected. HBV-prophylaxis strategy included NA with/without HBIG. HBV recurrence was defined as HBV deoxyribonucleic acid (DNA) positivity during the 1-year, post-LT follow-up. No HBV surface antibody titers were followed. A total of 103 patients with a median age of 60 years participated in the study. Hepatitis C virus was the most common etiology. Thirty-seven HBcAb negative recipients and 11 HBcAb positive recipients with undetectable HBV DNA received HBcAb positive organs and underwent prophylaxis with 4 doses of low-dose HBIG and NA. None of the recipients in our cohort had a recurrence of HBV at 1 year. Low-dose HBIG (1560 IU) × 4 days and NA, for HBcAb positive recipients and HBcAb positive donors, appear to be effective in preventing HBV reinfection during the post-LT period. Further trials are needed to confirm this observation.

Growth in open-source hardware designs combined with the low-cost of high performance optoelectronic and robotics components has supported a resurgence of in-house custom lab equipment development. We describe a low cost (below $700), open-source, fully customizable high-throughput imaging system for analytical microbiology applications. The system comprises a Raspberry Pi camera mounted on an aluminium extrusion frame with 3D-printed joints controlled by an Arduino microcontroller running open-source Repetier Host Firmware. The camera position is controlled by simple G-code scripts supplied from a Raspberry Pi singleboard computer and allow customized time-lapse imaging of microdevices over a large imaging area. Open-source OctoPrint software allows remote access and control. This simple yet effective design allows high-throughput microbiology testing in multiple formats including formats for bacterial motility, colony growth, microtitre plates and microfluidic devices termed 'lab-on-a-comb' to screen the effects of different culture media components and antibiotics on bacterial growth. The open-source robot design allows customization of the size of the imaging area; the current design has an imaging area of ~420 × 300mm, which allows 29 'lab-on-a-comb' devices to be imaged which is equivalent 3480 individual 1μl samples. The system can also be modified for fluorescence detection using LED and emission filters embedded on the PiCam for more sensitive detection of bacterial growth using fluorescent dyes.

Antibiotic susceptibility testing is vital to tackle the emergence and spread of antimicrobial resistance. Inexpensive digital CMOS cameras can be converted into portable digital microscopes using 3D printed x-y-z stages. Microscopic examination of bacterial motility can rapidly detect the response of microbes to antibiotics to determine susceptibility. Here, we present a new simple microdevice-miniature microscope cell measurement system for multiplexed antibiotic susceptibility testing. The microdevice is made using melt-extruded plastic film strips containing ten parallel 0.2 mm diameter microcapillaries. Two different antibiotics, ceftazidime and gentamicin, were prepared in Mueller-Hinton agar (0.4%) to produce an antibiotic-loaded microdevice for simple sample addition. This combination was selected to closely match current standard methods for both antibiotic susceptibility testing and motility testing. Use of low agar concentration permits observation of motile bacteria responding to antibiotic exposure as they enter capillaries. This device fits onto the OpenFlexure 3D-printed digital microscope using a Raspberry Pi computer and v2 camera, avoiding need for expensive laboratory microscopes. This inexpensive and portable digital microscope platform had sufficient magnification to detect motile bacteria, yet wide enough field of view to monitor bacteria behavior as they entered antibiotic-loaded microcapillaries. The image quality was sufficient to detect how bacterial motility was inhibited by different concentrations of antibiotic. We conclude that a 3D-printed Raspberry Pi-based microscope combined with disposable microfluidic test strips permit rapid, easy-to-use bacterial motility detection, with potential for aiding detection of antibiotic resistance.

The early detection of antimicrobial resistance remains an essential step in the selection and optimization of antibiotic treatments. Phenotypic antibiotic susceptibility testing including the measurement of minimum inhibitory concentration (MIC) remains critical for surveillance and diagnostic testing. Limitations to current testing methods include bulky labware and laborious methods. Furthermore, the requirement of a single strain of bacteria to be isolated from samples prior to antibiotic susceptibility testing delays results. The mixture of bacteria present in a sample may also have an altered resistance profile to the individual strains, and so measuring the susceptibility of the mixtures of organisms found in some samples may be desirable. To enable simultaneous MIC and bacterial species detection in a simple and rapid miniaturized format, a 3D-printed frame was designed for a multi-sample millifluidic dip-slide device that combines panels of identification culture media with a range of antibiotics (Ampicillin, Amoxicillin, Amikacin, Ceftazidime, Cefotaxime, Ofloxacin, Oxytetracycline, Streptomycin, Gentamycin and Imipenem) diluted in Muëller–Hinton Agar. Our proof-of-concept evaluation confirmed that the direct detection of more than one bacterium parallel to measuring MIC in samples is possible, which is validated using reference strains E. coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, Pseudomonas aeruginosa ATCC 10145, and Staphylococcus aureus ATCC 12600 and with mastitis milk samples collected from Reading University Farm. When mixtures were tested, a MIC value was obtained that reflected the most resistant organism present (i.e., highest MIC), suggesting it may be possible to estimate a minimum effective antibiotic concentration for mixtures directly from samples containing multiple pathogens. We conclude that this simple miniaturized approach to the rapid simultaneous identification and antibiotic susceptibility testing may be suitable for directly testing agricultural samples, which is achieved through shrinking conventional tests into a simple “dip-and-incubate” device that can be 3D printed anywhere.