Connexin hemichannels drive lactation-induced osteocyte acidification and perilacunar-canalicular remodeling.

The maternal skeleton experiences significant bone loss during lactation, followed by rapid restoration post weaning. Parathyroid-related protein (PTHrP)-induced acidification of the perilacunar matrix by osteocytes is crucial in this process, yet its mechanism remains unclear. Here, we identify Cx43 hemichannels (HCs) as key mediators of osteocyte acidification and perilacunar-canalicular remodeling (PLR). Utilizing transgenic mouse models expressing dominant-negative Cx43 mutants, we show that mice with impaired Cx43 HCs exhibit attenuated lactation-induced responses compared to wild-type and only gap junction-impaired groups, including lacunar enlargement, upregulation of PLR genes, and bone loss with compromised mechanical properties. Furthermore, inhibition of HCs by a Cx43 antibody blunts PTHrP-induced calcium influx and protein kinase A activation, followed by impaired osteocyte acidification. Additionally, impeded HCs suppress bone recovery during the post-lactation period. Our findings highlight the pivotal role of Cx43 HCs in orchestrating dynamic bone changes during lactation and recovery by regulating acidification and remodeling enzyme expression.

A Semiautonomous Deep Learning System to Reduce False Positives in Screening Mammography.

Purpose To evaluate the ability of a semiautonomous artificial intelligence (AI) model to identify screening mammograms not suspicious for breast cancer and reduce the number of false-positive examinations. Materials and Methods The deep learning algorithm was trained using 123 248 two-dimensional digital mammograms (6161 cancers) and a retrospective study was performed on three nonoverlapping datasets of 14 831 screening mammography examinations (1026 cancers) from two U.S. institutions and one U.K. institution (2008-2017). The stand-alone performance of humans and AI was compared. Human plus AI performance was simulated to examine reductions in the cancer detection rate, number of examinations, false-positive callbacks, and benign biopsies. Metrics were adjusted to mimic the natural distribution of a screening population, and bootstrapped CIs and P values were calculated. Results Retrospective evaluation on all datasets showed minimal changes to the cancer detection rate with use of the AI device (noninferiority margin of 0.25 cancers per 1000 examinations: U.S. dataset 1, P = .02; U.S. dataset 2, P < .001; U.K. dataset, P < .001). On U.S. dataset 1 (11 592 mammograms; 101 cancers; 3810 female patients; mean age, 57.3 years ± 10.0 [SD]), the device reduced screening examinations requiring radiologist interpretation by 41.6% (95% CI: 40.6%, 42.4%; P < .001), diagnostic examinations callbacks by 31.1% (95% CI: 28.7%, 33.4%; P < .001), and benign needle biopsies by 7.4% (95% CI: 4.1%, 12.4%; P < .001). U.S. dataset 2 (1362 mammograms; 330 cancers; 1293 female patients; mean age, 55.4 years ± 10.5) was reduced by 19.5% (95% CI: 16.9%, 22.1%; P < .001), 11.9% (95% CI: 8.6%, 15.7%; P < .001), and 6.5% (95% CI: 0.0%, 19.0%; P = .08), respectively. The U.K. dataset (1877 mammograms; 595 cancers; 1491 female patients; mean age, 63.5 years ± 7.1) was reduced by 36.8% (95% CI: 34.4%, 39.7%; P < .001), 17.1% (95% CI: 5.9%, 30.1%: P < .001), and 5.9% (95% CI: 2.9%, 11.5%; P < .001), respectively. Conclusion This work demonstrates the potential of a semiautonomous breast cancer screening system to reduce false positives, unnecessary procedures, patient anxiety, and medical expenses. Keywords: Artificial Intelligence, Semiautonomous Deep Learning, Breast Cancer, Screening Mammography Supplemental material is available for this article. Published under a CC BY 4.0 license.

Enhanced Calvarial Bone Repair Using ASCs Engineered with RNA-Guided Split dCas12a System that Co-Activates Sox 5, Sox6, and Long Non-Coding RNA H19.

Healing of large calvarial bone defects remains challenging. An RNA-guided Split dCas12a system is previously harnessed to activate long non-coding RNA H19 (lncRNA H19, referred to as H19 thereafter) in bone marrow-derived mesenchymal stem cells (BMSCs). H19 activation in BMSCs induces chondrogenic differentiation, switches bone healing pathways, and improves calvarial bone repair. Since adipose-derived stem cells (ASCs) can be harvested more easily in large quantity, here it is aimed to use ASCs as an alternative cell source. However, H19 activation alone using the Split dCas12a system in ASCs failed to elicit evident chondrogenesis. Therefore, split dCas12a activators are designed more to co-activate other chondroinductive transcription factors (Sox5, Sox6, and Sox9) to synergistically potentiate differentiation. It is found that co-activation of H19/Sox5/Sox6 in ASCs elicited more potent chondrogenic differentiation than activation of Sox5/Sox6/Sox9 or H19 alone. Co-activating H19/Sox5/Sox6 in ASCs significantly augmented in vitro cartilage formation and in vivo calvarial bone healing. These data altogether implicated the potentials of the Split dCas12a system to trigger multiplexed gene activation in ASCs for differentiation pathway reprogramming and tissue regeneration.

Racial, Ethnic, and Gender Differences in the Timing of Initiating the HPV Vaccine in the United States: the Case of Southeast Asian Americans.

Despite the availability of the human papillomavirus (HPV) vaccine, non-Latinx (NL) Southeast Asian Americans have the highest incidence of HPV-associated cervical cancer in the US. Little is known about NL-Southeast Asian Americans' HPV vaccination coverage due to being categorized under the "Asian American" monolith. Therefore, this study uses restricted data from the 2011-2018 National Health and Nutrition Examination Survey (NHANES) to disaggregate NL-Southeast Asian Americans and compare this population's age-specific probabilities of initiating HPV vaccinations to two Asian American subgroups (NL-East Asian and NL-South Asian Americans) and NL-White, NL-Black, and Latinx Americans. Multinomial logistic regression models examine the differences in the timing of initiating the HPV vaccine series, late (ages 13-26) or never, relative to on-time vaccination (by age 12). NL-Southeast Asian Americans are significantly more likely to never vaccinate and to vaccinate late than NL-White, NL-Black, and Latinx Americans, relative to on-time vaccination. NL-Southeast Asian American boys/men are significantly more likely to never initiate the HPV vaccine than Latinx boys/men, relative to on-time vaccination. NL-Southeast Asian American girls/women are significantly more likely to never vaccinate and vaccinate late than NL-White, NL-Black, and Latinx girls/women, relative to on-time vaccination. There are significant gender differences in uptake among all racial and ethnic groups, except among NL-Southeast and NL-East Asian Americans. Disaggregated data on NL-Southeast Asian Americans helps scholars and public health officials uncover health disparities and improve health interventions. Targeted HPV vaccine promotion and services for this population are needed to mitigate current and future health disparities and promote health equity.

Combination Therapy with Gallium Protoporphyrin and Gallium Nitrate Exhibits Enhanced Antimicrobial Activity In Vitro and In Vivo against Methicillin-Resistant Staphylococcus aureus.

There is a major need for the development of new therapeutics to combat antibiotic-resistant Staphylococcus aureus. Recently, gallium (Ga)-based complexes have shown promising antimicrobial effects against various bacteria, including multidrug-resistant organisms, by targeting multiple heme/iron-dependent metabolic pathways. Among these, Ga protoporphyrin (GaPP) inhibits bacterial growth by targeting heme pathways, including aerobic respiration. Ga(NO3)3, an iron mimetic, disrupts elemental iron pathways. Here, we demonstrate the enhanced antimicrobial activity of the combination of GaPP and Ga(NO3)3 against methicillin-resistant S. aureus (MRSA) under iron-limited conditions, including small colony variants (SCV). This therapy demonstrated significant antimicrobial activity without inducing slow-growing SCV. We also observed that the combination of GaPP and Ga(NO3)3 inhibited the MRSA catalase but not above that seen with Ga(NO3)3 alone. Neither GaPP nor Ga(NO3)3 alone or their combination inhibited the dominant superoxide dismutase expressed (SodA) under the iron-limited conditions examined. Intranasal administration of the combination of the two compounds improved drug biodistribution in the lungs compared to intraperitoneal administration. In a murine MRSA lung infection model, we observed a significant increase in survival and decrease in MRSA lung CFUs in mice that received combination therapy with intranasal GaPP and Ga(NO3)3 compared to untreated control or mice receiving GaPP or Ga(NO3)3 alone. No drug-related toxicity was observed as assessed histologically in the spleen, lung, nasal cavity, and kidney for both single and repeated doses of 10 mg Ga /Kg of mice over 13 days. Our results strongly suggest that GaPP and Ga(NO3)3 in combination have excellent synergism and potential to be developed as a novel therapy for infections with S. aureus.

Split dCas12a activator for lncRNA H19 activation to enhance BMSC differentiation and promote calvarial bone healing.

Healing of large calvarial bone defects in adults is challenging. We previously showed that inducing chondrogenic differentiation of mesenchymal stem cells from bone marrow (BMSC) or adipose tissue (ASC) before implantation can switch the repair pathway and improve calvarial bone healing. Split dCas12a activator is a new CRISPR activation system comprising the amino (N) and carboxyl (C) fragments of dCas12a protein, each being fused with synthetic transcription activators at both termini. The split dCas12a activator was shown to induce programmable gene expression in cell lines. Here we exploited the split dCas12a activator to activate the expression of chondroinductive long non-coding RNA H19. We showed that co-expression of the split N- and C-fragments resulted in spontaneous dimerization, which elicited stronger activation of H19 than full-length dCas12a activator in rat BMSC and ASC. We further packaged the entire split dCas12a activator system (13.2 kb) into a hybrid baculovirus vector, which enhanced and prolonged H19 activation for at least 14 days in BMSC and ASC. The extended H19 activation elicited potent chondrogenic differentiation and inhibited adipogenesis. Consequently, the engineered BMSC promoted in vitro cartilage formation and augmented calvarial bone healing in rats. These data implicated the potentials of the split dCas12a activator for stem cell engineering and regenerative medicine.

Effectiveness on post-stroke hemiplegia in patients: electroacupuncture plus cycling electroacupuncture alone.

OBJECTIVE: To evaluate the effectiveness of cycling in combination with electroacupuncture in treatment of post-stroke hemiplegia patients at National Hospital of Acupuncture, Vietnam. METHODS: The study was designed as a single-centre, outcome-assessor-blinded parallel randomised controlled trial with 120 post-stroke hemiplegia patients randomly assigned into two groups: electroacupuncture plus cycling (CT group) and electroacupuncture (AT group). Patients were assessed before and after the treatment (using muscle grading, modified Rankin, Barthel, Orgorozo scores and electromyography). Statistical Man-Whitney U test, and Fisher's exact tests were used to compare between CT and AT groups. RESULTS: The results reported statistically significant improvement in motor function in patients suffering from hemiplegia following ischemic stroke in both CT and AT groups. Patients in CT group experienced a greater improvement compared to those in AT group including better muscle contraction (increased frequency and amplitude of electromyography and increased muscle grading scale); increased recovery (Orgogozo scale), increased independency (Barthel scale) and decreased disability (Modified Rankin scale) (< 0.01). CONCLUSIONS: Combination with cycling training significantly improves the recovery of post-stroke patients treated with electroacupuncture.

Transplantation of Adipose-Tissue-Engineered Constructs with CRISPR-Mediated UCP1 Activation.

Thermogenic adipocytes have potential utility for the development of approaches to treat type 2 diabetes and obesity-associated diseases. Although several reports have proved the positive effect of beige and brown adipocyte transplantation in obese mice, translation to human cell therapy needs improvement. Here, we describe the application of CRISPR activation (CRISPRa) technology for generating safe and efficient adipose-tissue-engineered constructs with enhanced mitochondrial uncoupling protein 1 (UCP1) expression. We designed the CRISPRa system for the activation of UCP1 gene expression. CRISPRa-UCP1 was delivered into mature adipocytes by a baculovirus vector. Modified adipocytes were transplanted in C57BL/6 mice, followed by analysis of grafts, inflammation and systemic glucose metabolism. Staining of grafts on day 8 after transplantation shows them to contain UCP1-positive adipocytes. Following transplantation, adipocytes remain in grafts and exhibit expression of PGC1α transcription factor and hormone sensitive lipase (HSL). Transplantation of CRISPRa-UCP1-modified adipocytes does not influence glucose metabolism or inflammation in recipient mice. We show the utility and safety of baculovirus vectors for CRISPRa-based thermogenic gene activation. Our findings suggest a means of improving existing cell therapy approaches using baculovirus vectors and CRISPRa for modification and transplantation of non-immunogenic adipocytes.

Potent universal beta-coronavirus therapeutic activity mediated by direct respiratory administration of a Spike S2 domain-specific human neutralizing monoclonal antibody.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) marks the third novel ß-coronavirus to cause significant human mortality in the last two decades. Although vaccines are available, too few have been administered worldwide to keep the virus in check and to prevent mutations leading to immune escape. To determine if antibodies could be identified with universal coronavirus activity, plasma from convalescent subjects was screened for IgG against a stabilized pre-fusion SARS-CoV-2 spike S2 domain, which is highly conserved between human ß-coronavirus. From these subjects, several S2-specific human monoclonal antibodies (hmAbs) were developed that neutralized SARS-CoV-2 with recognition of all variants of concern (VoC) tested (Beta, Gamma, Delta, Epsilon, and Omicron). The hmAb 1249A8 emerged as the most potent and broad hmAb, able to recognize all human ß-coronavirus and neutralize SARS-CoV and MERS-CoV. 1249A8 demonstrated significant prophylactic activity in K18 hACE2 mice infected with SARS-CoV-2 lineage A and lineage B Beta, and Omicron VoC. 1249A8 delivered as a single 4 mg/kg intranasal (i.n.) dose to hamsters 12 hours following infection with SARS-CoV-2 Delta protected them from weight loss, with therapeutic activity further enhanced when combined with 1213H7, an S1-specific neutralizing hmAb. As little as 2 mg/kg of 1249A8 i.n. dose 12 hours following infection with SARS-CoV Urbani strain, protected hamsters from weight loss and significantly reduced upper and lower respiratory viral burden. These results indicate in vivo cooperativity between S1 and S2 specific neutralizing hmAbs and that potent universal coronavirus neutralizing mAbs with therapeutic potential can be induced in humans and can guide universal coronavirus vaccine development.

The First Case of Coxiella Burnetti Infection Detected Through Bone Marrow Biopsy in Vietnam.

Coxiella burnetii is an obligate intracellular bacterium that causes the zoonotic infectious disease, Q fever. The common clinical presentation is fever, hepatitis, and pneumonia; laboratory examination could reveal pancytopenia, elevated liver enzymes. In bone marrow, many fibrin ring granulomas, also known as "Doughnut" granulomas can be seen and suggest the diagnosis of Q fever. However, these bone marrow granulomas can also be presented in infectious diseases by other pathogens such as EBV, CMV, and HBV; therefore, other serology or PCR-based tests are needed to confirm the diagnosis of Q fever. We report the first case of acute Q fever in Vietnam, presented as a fever of unknown origin with hepatitis in a 53-year-old male patient. A bone marrow biopsy was performed and showed various fibrin ring granulomas; therefore, Coxiella was suspected and the diagnosis was confirmed by PCR. Some infectious diseases can cause specific changes in the bone marrow, such as Doughnut granulomas in Q fever. These features can help direct the diagnosis and decide earlier treatment for the patient.

Potent universal-coronavirus therapeutic activity mediated by direct respiratory administration of a Spike S2 domain-specific human neutralizing monoclonal antibody.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) marks the third novel ß-coronavirus to cause significant human mortality in the last two decades. Although vaccines are available, too few have been administered worldwide to keep the virus in check and to prevent mutations leading to immune escape. To determine if antibodies could be identified with universal coronavirus activity, plasma from convalescent subjects was screened for IgG against a stabilized pre-fusion SARS-CoV-2 spike S2 domain, which is highly conserved between human ß-coronavirus. From these subjects, several S2-specific human monoclonal antibodies (hmAbs) were developed that neutralized SARS-CoV-2 with recognition of all variants of concern (VoC) tested (Beta, Gamma, Delta, Epsilon, and Omicron). The hmAb 1249A8 emerged as the most potent and broad hmAb, able to recognize all human ß-coronavirus and neutralize SARS-CoV and MERS-CoV. 1249A8 demonstrated significant prophylactic activity in K18 hACE2 mice infected with SARS-CoV-2 lineage A and lineage B Beta, and Omicron VoC. 1249A8 delivered as a single 4 mg/kg intranasal (i.n.) dose to hamsters 12 hours following infection with SARS-CoV-2 Delta protected them from weight loss, with therapeutic activity further enhanced when combined with 1213H7, an S1-specific neutralizing hmAb. As little as 2 mg/kg of 1249A8 i.n. dose 12 hours following infection with SARS-CoV Urbani strain, protected hamsters from weight loss and significantly reduced upper and lower respiratory viral burden. These results indicate in vivo cooperativity between S1 and S2 specific neutralizing hmAbs and that potent universal coronavirus neutralizing mAbs with therapeutic potential can be induced in humans and can guide universal coronavirus vaccine development.

Bi-directional gene activation and repression promote ASC differentiation and enhance bone healing in osteoporotic rats.

Calvarial bone healing is challenging, especially for individuals with osteoporosis because stem cells from osteoporotic patients are highly prone to adipogenic differentiation. Based on previous findings that chondrogenic induction of adipose-derived stem cells (ASCs) can augment calvarial bone healing, we hypothesized that activating chondroinductive Sox Trio genes (Sox5, Sox6, Sox9) and repressing adipoinductive genes (C/ebp-α, Ppar-γ) in osteoporotic ASCs can reprogram cell differentiation and improve calvarial bone healing after implantation. However, simultaneous gene activation and repression in ASCs is difficult. To tackle this problem, we built a CRISPR-BiD system for bi-directional gene regulation. Specifically, we built a CRISPR-AceTran system that exploited both histone acetylation and transcription activation for synergistic Sox Trio activation. We also developed a CRISPR interference (CRISPRi) system that exploited DNA methylation for repression of adipoinductive genes. We combined CRISPR-AceTran and CRISPRi to form the CRISPR-BiD system, which harnessed three mechanisms (transcription activation, histone acetylation, and DNA methylation). After delivery into osteoporotic rat ASCs, CRISPR-BiD significantly enhanced chondrogenesis and in vitro cartilage formation. Implantation of the engineered osteoporotic ASCs into critical-sized calvarial bone defects significantly improved bone healing in osteoporotic rats. These results implicated the potential of the CRISPR-BiD system for bi-directional regulation of cell fate and regenerative medicine.

On-time, late, or never: Incorporating intersectionality to predict age-specific probabilities of initiating the HPV vaccine series.

The HPV vaccine's effectiveness in protecting against the human papillomavirus (HPV) is dependent on both the timing of vaccine initiation (by age 12) and number of vaccine doses. This study used an intersectional approach to examine how gender and its intersection with race/ethnicity predict age-specific probabilities of initiating HPV vaccinations for boys/men and girls/women across four racial/ethnic groups: non-Latinx (NL) white, NL-Black, Latinx, and NL-Asian American. Multinomial logistic regression models-with on-time vaccination as the base outcome-examined the timing of initiating HPV vaccinations (never or late) in the 2011-2016 National Health and Nutrition Examination Survey (NHANES). This study finds that the multiplicative interaction term between gender and race/ethnicity produces significant results in the likelihood of initiating HPV vaccine uptake: NL-Asian American boys/men have the highest risk of not vaccinating on time. This finding would have been hidden with a one-dimensional analysis.

Anti-inflammatory norclerodane diterpenoids and tetrahydrophenanthrene from the leaves and stems of Dioscorea bulbifera.

Chemical investigation of the leaves and stems of Dioscorea bulbifera resulted in isolation of 10 compounds, including three new norclerodane diterpenoids, diosbulbiferins A (1) and B (2) and diosbulbiferinoside A (3), and one new natural congener, diosbulbiferin C (4), along with one new tetrahydrophenanthrene, diosbulbinone (8). Their structures were elucidated by comprehensive analyses of spectroscopic methods, including NMR and mass spectra. The absolute configurations of compounds 1-3 and 8 were deduced by time-dependent density functional theory (TD-DFT) electronic circular dichroism (ECD) spectroscopic analyses. In addition, cytotoxic effects against MCF-7, HepG2, and SK-Mel-2 cancer cells and in vitro anti-inflammatory effects of the isolated compounds in LPS-stimulated BV2 microglial cells were also reported.

CRISPR activation of long non-coding RNA DANCR promotes bone regeneration.

Healing of large calvarial bone defects in adults adopts intramembranous pathway and is difficult. Implantation of adipose-derived stem cells (ASC) that differentiate towards chondrogenic lineage can switch the bone repair pathway and improve calvarial bone healing. Long non-coding RNA DANCR was recently uncovered to promote chondrogenesis, but its roles in rat ASC (rASC) chondrogenesis and bone healing stimulation have yet to be explored. Here we first verified that DANCR expression promoted rASC chondrogenesis, thus we harnessed CRISPR activation (CRISPRa) technology to upregulate endogenous DANCR, stimulate rASC chondrogenesis and improve calvarial bone healing in rats. We generated 4 different dCas9-VPR orthologues by fusing a tripartite transcription activator domain VPR to catalytically dead Cas9 (dCas9) derived from 4 different bacteria, and compared the degree of activation using the 4 different dCas9-VPR. We unveiled surprisingly that the most commonly used dCas9-VPR derived from Streptococcus pyogenes barely activated DANCR. Nonetheless dCas9-VPR from Staphylococcus aureus (SadCas9-VPR) triggered efficient activation of DANCR in rASC. Delivery of SadCas9-VPR and the associated guide RNA into rASC substantially enhanced chondrogenic differentiation of rASC and augmented cartilage formation in vitro. Implantation of the engineered rASC remarkably potentiated the calvarial bone healing in rats. Furthermore, we identified that DANCR improved the rASC chondrogenesis through inhibition of miR-203a and miR-214. These results collectively proved that DANCR activation by SadCas9-VPR-based CRISPRa provides a novel therapeutic approach to improving calvarial bone healing.

Adolescent Technology, Sleep, and Physical Activity Time in Two US Cohorts.

The advent of internet-enabled mobile digital devices has transformed US adolescent technology use over the last decade, yet little is known about how these changes map onto other health-related behaviors. We provide a national profile of how contemporary technology use fits into adolescents' daily health lifestyles compared to the previous generation, with particular attention to whether and for whom technology use displaces time spent in sleep or physical activity. Time diaries were collected from 11-17 year olds in 2002-03 (N=1,139) and 2014-16 (N=527) through the US Panel Study of Income Dynamics Child Development Supplement. Contemporary adolescents spent 40 minutes more per week in technology-focused activities, but their composition was more varied compared to the earlier cohort. Contemporary technology use was predictive of less time in physical activity, and adolescents engaged in frequent video game play spent less time in physical activity compared to peers with other technology use profiles.

Therapeutic activity of an inhaled potent SARS-CoV-2 neutralizing human monoclonal antibody in hamsters.

SARS-CoV-2 infection results in viral burden in the respiratory tract, enabling transmission and leading to substantial lung pathology. The 1212C2 fully human monoclonal antibody was derived from an IgM memory B cell of a COVID-19 patient, has high affinity for the Spike protein receptor binding domain, neutralizes SARS-CoV-2, and exhibits in vivo prophylactic and therapeutic activity in hamsters when delivered intraperitoneally, reducing upper and lower respiratory viral burden and lung pathology. Inhalation of nebulized 1212C2 at levels as low as 0.6 mg/kg, corresponding to 0.03 mg/kg lung-deposited dose, reduced the viral burden below the detection limit and mitigated lung pathology. The therapeutic efficacy of an exceedingly low dose of inhaled 1212C2 supports the rationale for local lung delivery for dose-sparing benefits, as compared to the conventional parenteral route of administration. These results suggest that the clinical development of 1212C2 formulated and delivered via inhalation for the treatment of SARS-CoV-2 infection should be considered.

CRISPR interference-mediated noggin knockdown promotes BMP2-induced osteogenesis and calvarial bone healing.

Healing of large calvarial bone defects remains a challenging task in the clinical setting. Although BMP2 (bone morphogenetic protein 2) is a potent growth factor that can induce bone repair, BMP2 provokes the expression of antagonist Noggin that self-restricts its bioactivity. CRISPR interference (CRISPRi) is a technology for programmable gene suppression but its application in regenerative medicine is still in its infancy. We reasoned that Nog inhibition, concurrent with BMP2 overexpression, can promote the osteogenesis of adipose-derived stem cells (ASC) and improve calvarial bone healing. We designed and exploited a hybrid baculovirus (BV) system for the delivery of BMP2 gene and CRISPRi system targeting Nog. After BV-mediated co-delivery into ASC, the system conferred prolonged BMP2 expression and stimulated Nog expression while the CRISPRi system effectively repressed Nog upregulation for at least 14 days. The CRISPRi-mediated Nog knockdown, along with BMP2 overexpression, additively stimulated the osteogenic differentiation of ASC. Implantation of the CRISPRi-engineered ASC into the critical size defects at the calvaria significantly enhanced the calvarial bone healing and matrix mineralization. These data altogether implicate the potentials of CRISPRi-mediated gene knockdown for cell fate regulation and tissue regeneration.

Emerging Role of Circulating Tumor Cells in Gastric Cancer.

With over 1 million incidence cases and more than 780,000 deaths in 2018, gastric cancer (GC) was ranked as the 5th most common cancer and the 3rd leading cause of cancer deaths worldwide. Though several biomarkers, including carcinoembryonic antigen (CEA), cancer antigen 19-9 (CA19-9), and cancer antigen 72-4 (CA72-4), have been identified, their diagnostic accuracies were modest. Circulating tumor cells (CTCs), cells derived from tumors and present in body fluids, have recently emerged as promising biomarkers, diagnostically and prognostically, of cancers, including GC. In this review, we present the landscape of CTCs from migration, to the presence in circulation, biologic properties, and morphologic heterogeneities. We evaluated clinical implications of CTCs in GC patients, including diagnosis, prognosis, and therapeutic management, as well as their application in immunotherapy. On the one hand, major challenges in using CTCs in GC were analyzed, from the differences of cut-off values of CTC positivity, to techniques used for sampling, storage conditions, and CTC molecular markers, as well as the unavailability of relevant enrichment and detection techniques. On the other hand, we discussed future perspectives of using CTCs in GC management and research, including the use of circulating tumor microembolies; of CTC checkpoint blockade in immunotherapy; and of organoid models. Despite the fact that there are remaining challenges in techniques, CTCs have potential as novel biomarkers and/or a non-invasive method for diagnostics, prognostics, and treatment monitoring of GC, particularly in the era of precision medicine.

Selective aggregation by ultrasonic standing waves through gas nuclei on the particle surface.

Gas nuclei in water are usually too small to be directly observed. They will grow into bubbles under the negative pressure, which is called cavitation (heterogeneous cavitation). In this study, the gas nuclei in the hydrophilic and hydrophobic silica particle suspension were investigated using the transient cavitation threshold measured by a high-intensity focused ultrasound (HIFU). The transient cavitation bubbles were also observed by a high-speed camera. The results showed that the nuclei only exist on the surface of hydrophobic particles. Furthermore, the aggregation experiments revealed that the aggregates were only formed in the hydrophobic silica suspension by ultrasonic standing waves (USW) at 200 kHz. This distinct difference was mainly due to the formation of gas nuclei on hydrophobic silica particles, which grew and coalesced into stable bubbles under the 200 kHz USW. The aggregation process in suspension was observed by a CCD camera. Moreover, the cavitation thresholds and acoustic radiation forces were analyzed to explain the mechanism of the acoustic aggregation. This study showed a very promising acoustic method for the selective aggregation of hydrophobic particles, which might be efficiently used in the mineral separation industry.