ACE2 acts as a novel regulator of TMPRSS2-catalyzed proteolytic activation of influenza A virus in airway cells.

The transmembrane serine protease 2 (TMPRSS2) activates the outer structural proteins of a number of respiratory viruses including influenza A virus (IAV), parainfluenza viruses, and various coronaviruses for membrane fusion. Previous studies showed that TMPRSS2 interacts with the carboxypeptidase angiotensin-converting enzyme 2 (ACE2), a cell surface protein that serves as an entry receptor for some coronaviruses. Here, by using protease activity assays, we determine that ACE2 increases the enzymatic activity of TMPRSS2 in a non-catalytic manner. Furthermore, we demonstrate that ACE2 knockdown inhibits TMPRSS2-mediated cleavage of IAV hemagglutinin (HA) in Calu-3 human airway cells and suppresses virus titers 100- to 1.000-fold. Transient expression of ACE2 in ACE2-deficient cells increased TMPRSS2-mediated HA cleavage and IAV replication. ACE2 knockdown also reduced titers of MERS-CoV and prevented S cleavage by TMPRSS2 in Calu-3 cells. By contrast, proteolytic activation and multicycle replication of IAV with multibasic HA cleavage site typically cleaved by furin were not affected by ACE2 knockdown. Co-immunoprecipitation analysis revealed that ACE2-TMPRSS2 interaction requires the enzymatic activity of TMPRSS2 and the carboxypeptidase domain of ACE2. Together, our data identify ACE2 as a new co-factor or stabilizer of TMPRSS2 activity and as a novel host cell factor involved in proteolytic activation and spread of IAV in human airway cells. Furthermore, our data indicate that ACE2 is involved in the TMPRSS2-catalyzed activation of additional respiratory viruses including MERS-CoV.IMPORTANCEProteolytic cleavage of viral envelope proteins by host cell proteases is essential for the infectivity of many viruses and relevant proteases provide promising drug targets. The transmembrane serine protease 2 (TMPRSS2) has been identified as a major activating protease of several respiratory viruses, including influenza A virus. TMPRSS2 was previously shown to interact with angiotensin-converting enzyme 2 (ACE2). Here, we report the mechanistic details of this interaction. We demonstrate that ACE2 increases or stabilizes the enzymatic activity of TMPRSS2. Furthermore, we describe ACE2 involvement in TMPRSS2-catalyzed cleavage of the influenza A virus hemagglutinin and MERS-CoV spike protein in human airway cells. These findings expand our knowledge of the activation of respiratory viruses by TMPRSS2 and the host cell factors involved. In addition, our results could help to elucidate a physiological role for TMPRSS2.

Development of DG9 peptide-conjugated single- and multi-exon skipping therapies for the treatment of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is primarily caused by out-of-frame deletions in the dystrophin gene. Exon skipping using phosphorodiamidate morpholino oligomers (PMOs) converts out-of-frame to in-frame mutations, producing partially functional dystrophin. Four single-exon skipping PMOs are approved for DMD but treat only 8 to 14% of patients each, and some exhibit poor efficacy. Alternatively, exons 45 to 55 skipping could treat 40 to 47% of all patients and is associated with improved clinical outcomes. Here, we report the development of peptide-conjugated PMOs for exons 45 to 55 skipping. Experiments with immortalized patient myotubes revealed that exons 45 to 55 could be skipped by targeting as few as five exons. We also found that conjugating DG9, a cell-penetrating peptide, to PMOs improved single-exon 51 skipping, dystrophin restoration, and muscle function in hDMDdel52;mdx mice. Local administration of a minimized exons 45 to 55-skipping DG9-PMO mixture restored dystrophin production. This study provides proof of concept toward the development of a more economical and effective exons 45 to 55-skipping DMD therapy.

Interpretable CT radiomics model for invasiveness prediction in patients with ground-glass nodules.

AIM: To evaluate the performance of an interpretable computed tomography (CT) radiomic model in predicting the invasiveness of ground-glass nodules (GGNs). MATERIALS AND METHODS: The study was conducted retrospectively from 1 August 2017 to 1 August 2022, at three different centres. Two hundred and thirty patients with GGNs were enrolled at centre I as a training cohort. Centres II (n=157) and III (n=156) formed two external validation cohorts. Radiomics features extracted based on CT were reduced by a coarse-fine feature screening strategy. A radiomic model was developed through the use of the LASSO (least absolute shrinkage and selection operator) and XGBoost algorithms. Then, a radiological model was established through multivariate logistic regression analysis. Finally, the interpretability of the model was explored using SHapley Additive exPlanations (SHAP). RESULTS: The radiomic XGBoost model outperformed the radiomic logistic model and radiological model in assessing the invasiveness of GGNs. The area under the curve (AUC) values for the radiomic XGBoost model were 0.885 (95% confidence interval [CI] 0.836-0.923), 0.853 (95% CI 0.790-0.906), and 0.838 (95% CI 0.773-0.902) in the training and the two external validation cohorts, respectively. The SHAP method allowed for both a quantitative and visual representation of how decisions were made using a given model for each individual patient. This can provide a deeper understanding of the decision-making mechanisms within the model and the factors that contribute to its prediction effectiveness. CONCLUSIONS: The present interpretable CT radiomics model has the potential to preoperatively evaluate the invasiveness of GGNs. Furthermore, it can provide personalised, image-based clinical-decision support.

Mirror Syndrome Combined with Postpartum Hemolytic Uremic Syndrome.

BACKGROUND: Mirror syndrome is a rare disease characterized by "triple edema", while Hemolytic Uremic Syndrome (PHUS) is a serious disease that occurs within a short period of time after the end of pregnancy, with a low prevalence and poor prognosis, and it is even rarer for both to occur in the same patient. METHODS: We report a case of mirror syndrome combined with PHUS and analyze the clinical data to improve the understanding of the disease. RESULTS: The patient presented clinically with "triple edema" and was diagnosed with mirror image syndrome. After cesarean section, the patient developed cardiac insufficiency, renal insufficiency, hemolysis, and other symptoms and was diagnosed as PHUS. After active treatment, the maternal prognosis was good. CONCLUSIONS: Mirror syndrome and PHUS are both clinically rare diseases with poor long-term prognosis if not diagnosed and treated in a timely manner; therefore, awareness of the diseases, early and accurate diagnosis and timely and correct treatment should be improved.

Influence of intracranial hemorrhage on clinical outcome in acute vertebrobasilar artery occlusion undergoing endovascular treatment.

BACKGROUND AND PURPOSE: The effect of intracranial hemorrhage (ICH) on the outcome of patients with large-vessel occlusion undergoing endovascular treatment (EVT) has mainly focused on the anterior circulation. Knowledge of the relationship between ICH and outcomes in patients with acute vertebrobasilar artery occlusion (VBAO) receiving EVT is limited. We aimed to assess whether ICH is a prognostic marker for acute VBAO following EVT. METHODS: Patients who underwent EVT for acute VBAO in the acute posterior circulation ischemic stroke (PERSIST) registry were included. All patients were classified as having no or any-ICH. Any-ICH was subdivided into asymptomatic and symptomatic ICH. A multivariate regression analysis was performed to evaluate the association between ICH and functional outcomes in patients with acute VBAO after receiving EVT. RESULTS: Five hundred and forty-seven patients, including 107 patients with ICH (19.6%): 38 (7.0%) and 69 (12.6%) with symptomatic and asymptomatic ICH, respectively. After adjustment for potential confounders, any-ICH was independently associated with reduced chance of favorable outcome (OR 0.39, 95% CI 0.21-0.72, P=0.003), functional independence (OR 0.24, 95% CI 0.16-0.52, P<0.001), and excellent outcome (OR 0.34, 95% CI 0.15-0.75, P=0.008), and increased mortality risk (OR 2.14, 95% CI 1.30-3.51, P=0.003). Symptomatic ICH had a similar association. Moreover, asymptomatic ICH was a negative predictor of functional independence (OR 0.39, 95% CI 0.17-0.88, P=0.024). CONCLUSION: Any- and symptomatic ICH were strongly associated with worse clinical outcomes and increased mortality in patients with acute VBAO who underwent EVT. Asymptomatic ICH was an inverse predictor of functional independence.

[Gastric hamartomatous inverted polyps: a clinicopathological analysis of five cases].

Objective: To investigate the endoscopic and histopathological features, diagnosis and differential diagnosis of gastric hamartomatous inverted polyp (GHIP). Methods: Five cases of GHIP were collected at the University Town Hospital of Guangdong Provincial Hospital of Chinese Medicine, Guangzhou, China, from May 2021 to May 2023. The endoscopic, pathological and immunohistochemical features of the 5 GHIP cases were analyzed. The relevant literature was reviewed. Results: There were 3 males and 2 females, aged from 49 to 60 years, with a mean age of 56 years. The lesions were located in the fundus and body of the stomach, and presented as polyps or masses under endoscopy. Microscopically, the lesions were mainly in the submucosa and consisted of lobulated or clustered gastric glandular epithelium surrounded by hyperplastic smooth muscle. In some areas, there were differentiated glandular elements mimicking the normal gastric mucosa. The irregularly dilated glandular elements in the center were lined by hyperplastic foveolar epithelium, while the glands in the periphery were fundic or pyloric glands. In addition, in some areas, the glands showed cystic expansion, disordered arrangement and lack of differentiation. The hyperplastic glandular epithelium included foveolar epithelium, fundic gland and pyloric gland. There were scattered neuroendocrine cells and smooth muscle bundles in the stroma. Immunohistochemically, the tumor cells were positive for MUC5AC, MUC6, Pepsinogen Ⅰ and H+/K+ ATPase ß, but negative for MUC2. The scattered neuroendocrine cells were positive for synaptophysin, and the desmin stain highlighted hyperplastic smooth muscle bundles. One case was classified as type 2 gastric inverted polyp, and 4 cases were classified as type 3. Conclusions: GHIP is a rare gastric polyp with unique histological features. It should be distinguished from inverted hyperplastic polyp, gastritis cystica profunda, adenomyoma, hyperplastic polyps and well-differentiated gastric tubular adenocarcinoma, etc. Improving the understanding of its pathogenesis and diagnostic features can help avoid misdiagnoses.

Unanchored K48-linked polyubiquitin synthesized by the E3-ubiquitin ligase TRIM6 stimulates the interferon-IKKε kinase-mediated antiviral response.

Type I interferons (IFN-I) are essential antiviral cytokines produced upon microbial infection. IFN-I elicits this activity through the upregulation of hundreds of IFN-I-stimulated genes (ISGs). The full breadth of ISG induction demands activation of a number of cellular factors including the IκB kinase epsilon (IKKε). However, the mechanism of IKKε activation upon IFN receptor signaling has remained elusive. Here we show that TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family of proteins, interacted with IKKε and promoted induction of IKKε-dependent ISGs. TRIM6 and the E2-ubiquitin conjugase UbE2K cooperated in the synthesis of unanchored K48-linked polyubiquitin chains, which activated IKKε for subsequent STAT1 phosphorylation. Our work attributes a previously unrecognized activating role of K48-linked unanchored polyubiquitin chains in kinase activation and identifies the UbE2K-TRIM6-ubiquitin axis as critical for IFN signaling and antiviral response.

Cell-penetrating peptide-conjugated Morpholino rescues SMA in a symptomatic preclinical model.

Spinal muscular atrophy (SMA) is a motor neuron disease and the leading genetic cause of infant mortality. Recently approved SMA therapies have transformed a deadly disease into a survivable one, but these compounds show a wide spectrum of clinical response and effective rescue only in the early stages of the disease. Therefore, safe, symptomatic-suitable, non-invasive treatments with high clinical impact across different phenotypes are urgently needed. We conjugated antisense oligonucleotides with Morpholino (MO) chemistry, which increase SMN protein levels, to cell-penetrating peptides (CPPs) for better cellular distribution. Systemically administered MOs linked to r6 and (RXRRBR)2XB peptides crossed the blood-brain barrier and increased SMN protein levels remarkably, causing striking improvement of survival, neuromuscular function, and neuropathology, even in symptomatic SMA animals. Our study demonstrates that MO-CPP conjugates can significantly expand the therapeutic window through minimally invasive systemic administration, opening the path for clinical applications of this strategy.

Enhanced delivery of peptide-morpholino oligonucleotides with a small molecule to correct splicing defects in the lung.

Pulmonary diseases offer many targets for oligonucleotide therapeutics. However, effective delivery of oligonucleotides to the lung is challenging. For example, splicing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) affect a significant cohort of Cystic Fibrosis (CF) patients. These individuals could potentially benefit from treatment with splice switching oligonucleotides (SSOs) that can modulate splicing of CFTR and restore its activity. However, previous studies in cell culture used oligonucleotide transfection methods that cannot be safely translated in vivo. In this report, we demonstrate effective correction of a splicing mutation in the lung of a mouse model using SSOs. Moreover, we also demonstrate effective correction of a CFTR splicing mutation in a pre-clinical CF patient-derived cell model. We utilized a highly effective delivery strategy for oligonucleotides by combining peptide-morpholino (PPMO) SSOs with small molecules termed OECs. PPMOs distribute broadly into the lung and other tissues while OECs potentiate the effects of oligonucleotides by releasing them from endosomal entrapment. The combined PPMO plus OEC approach proved to be effective both in CF patient cells and in vivo in the mouse lung and thus may offer a path to the development of novel therapeutics for splicing mutations in CF and other lung diseases.

Simulating soil C dynamics under intensive agricultural systems and climate change scenarios in the Matopiba region, Brazil.

The recent agricultural expansion in the Matopiba region, Brazil's new agricultural frontier, has raised questions about the risk of increasing soil organic carbon (SOC) loss as large areas of native vegetation (NV; i.e., Cerrado biome) have been replaced by large-scale mechanized agriculture. Although sustainable managements, such as integrated crop-livestock (ICL) systems, are considered strategic to counterbalance the SOC loss associated with land-use change (LUC) while keeping food production, little is known about their long-term effects on SOC stocks in the Matopiba region. To this end, we used the DayCent model to simulate the effects of converting the management commonly used in this region, i.e., soybean-cotton rotation under no-tillage (NT), into ICL systems with distinct levels of intensification (e.g., crop rotations: soybean-pasture and soybean-pasture-cotton; soil and crop management: grass irrigation, scarification/harrowing, and length of grass cultivation) on long term SOC dynamics. Additionally, data from two projected climate scenarios: SSP2-4.5 [greenhouse gases emissions (GHG) will not change markedly over time and global temperature will increase by 2.0 °C by 2060] and SSP5-8.5 (marked changes in GHG emissions are expected to occur resulting in an increase of 2.4 and 4.4 °C in global temperature in the middle and at the end of the century) were included in our simulations to evaluate climate change effects on SOC dynamics in this region. Based on a 50-yr-time frame simulation, we observed that SOC stocks under ICL systems were, on average, 23% and 47% higher than in the NV (36.9 Mg ha-1) and soybean-cotton rotation under NT (30.9 Mg ha-1), respectively. Growing grasses interlaid with crops was crucial to increase SOC stocks even when disruptive soil practices were followed. Although the irrigation of grass resulted in an early increase of SOC stocks and a higher pasture stoking rate, it did not increase SOC stocks in the long term compared to non-irrigated treatments. The SSP2-4.5 and SSP5-8.5 climate scenarios had little effects on SOC dynamics in the simulated ICL systems. However, additional SOC loss (∼0.065 Mg ha-1 yr-1) is predicted to occur if the current management is not improved. These findings can help guide management decisions for the Matopiba region, Brazil, to alleviate the anthropogenic pressure associated with agriculture development. More broadly, they confirm that crop-livestock integration in croplands is a successful strategy to regenerate SOC.

[Regulation and influence of mitochondria on macrophages during hepatitis B virus infection].

Hepatitis B virus (HBV) infection is an important public health concern, as approximately 3.5% of the world's population is currently chronically infected. Chronic HBV infection is the primary cause of cirrhosis, hepatocellular carcinoma, and deaths related to liver disease globally. Studies have found that in HBV infection, viruses can directly or indirectly regulate mitochondrial energy metabolism, oxidative stress, respiratory chain metabolites, and autophagy, thereby altering macrophage activation status, differentiation types, and related cytokine secretion type and quantity regulations. Therefore, mitochondria have become an important signal source for macrophages to participate in the body's immune system during HBV infection, providing a basis for mitochondria to be considered as a potential therapeutic target for chronic hepatitis B.

CX3CR1 Is a Receptor for Human Respiratory Syncytial Virus in Cotton Rats.

Respiratory syncytial virus (RSV) has been reported to use CX3CR1 in vitro as a receptor on cultured primary human airway epithelial cultures. To evaluate CX3CR1 as the receptor for RSV in vivo, we used the cotton rat animal model because of its high permissiveness for RSV infection. Sequencing the cotton rat CX3CR1 gene revealed 91% amino acid similarity to human CX3CR1. Previous work found that RSV binds to CX3CR1 via its attachment glycoprotein (G protein) to infect primary human airway cultures. To determine whether CX3CR1-G protein interaction is necessary for RSV infection, recombinant RSVs containing mutations in the CX3CR1 binding site of the G protein were tested in cotton rats. In contrast to wild-type virus, viral mutants did not grow in the lungs of cotton rats. When RSV was incubated with an antibody blocking the CX3CR1 binding site of G protein and subsequently inoculated intranasally into cotton rats, no virus was found in the lungs 4 days postinfection. In contrast, growth of RSV was not affected after preincubation with heparan sulfate (the receptor for RSV on immortalized cell lines). A reduction in CX3CR1 expression in the cotton rat lung through the use of peptide-conjugated morpholino oligomers led to a 10-fold reduction in RSV titers at day 4 postinfection. In summary, these results indicate that CX3CR1 functions as a receptor for RSV in cotton rats and, in combination with data from human airway epithelial cell cultures, strongly suggest that CX3CR1 is a primary receptor for naturally acquired RSV infection. IMPORTANCE The knowledge about a virus receptor is useful to better understand the uptake of a virus into a cell and potentially develop antivirals directed against either the receptor molecule on the cell or the receptor-binding protein of the virus. Among a number of potential receptor proteins, human CX3CR1 has been demonstrated to act as a receptor for respiratory syncytial virus (RSV) on human epithelial cells in tissue culture. Here, we report that the cotton rat CX3CR1, which is similar to the human molecule, acts as a receptor in vivo. This study strengthens the argument that CX3CR1 is a receptor molecule for RSV.

The binding between NPM and H2B proteins signals for the diabetes-associated centrosome amplification.

Diabetes not only increases the risk for cancer but also promotes cancer metastasis. Centrosome amplification (CA) is sufficient to initiate tumorigenesis and can enhance the invasion potential of cancer cells. We have reported that diabetes can induce CA, with diabetic pathophysiological factors as the triggers, which involves the signaling of nucleophosmin (NPM). Thus, CA can serve as a candidate biological link between diabetes and cancer. In the present study, we attempted to identify the NPM binding partners and investigated whether the binding between NPM and its partner mediated the CA. We confirmed that high glucose, insulin, and palmitic acid cancer could elicit CA in the HCT16 colon cancer cells and found that the experimental treatment increased the binding between NPM and H2B, but not between p-NPM and H2B. The molecular docking analysis supported the fact that NPM and H2B could bind to each other through various amino acid residues. The treatment also increased the colocalization of NPM and H2B in the cytosol. Importantly, disruption of the NPM1-H2B complex by individual knockdown of the protein level of NPM or H2B led to the inhibition of the treatment-evoked CA. In conclusion, our results suggest that the binding between NPM and H2B proteins signals for the CA by high glucose, insulin, and palmitic acid.

Inhibition of SARS-CoV-2 in Vero cell cultures by peptide-conjugated morpholino oligomers.

BACKGROUND: As the causative agent of COVID-19, SARS-CoV-2 is a pathogen of immense importance to global public health. Development of innovative direct-acting antiviral agents is sorely needed to address this virus. Peptide-conjugated morpholino oligomers (PPMO) are antisense compounds composed of a phosphorodiamidate morpholino oligomer covalently conjugated to a cell-penetrating peptide. PPMO require no delivery assistance to enter cells and are able to reduce expression of targeted RNA through sequence-specific steric blocking. METHODS: Five PPMO designed against sequences of genomic RNA in the SARS-CoV-2 5'-untranslated region and a negative control PPMO of random sequence were synthesized. Each PPMO was evaluated for its effect on the viability of uninfected cells and its inhibitory effect on the replication of SARS-CoV-2 in Vero-E6 cell cultures. Cell viability was evaluated with an ATP-based method using a 48 h PPMO treatment time. Viral growth was measured with quantitative RT-PCR and TCID50 infectivity assays from experiments where cells received a 5 h PPMO treatment time. RESULTS: PPMO designed to base-pair with sequence in the 5' terminal region or the leader transcription regulatory sequence region of SARS-CoV-2 genomic RNA were highly efficacious, reducing viral titres by up to 4-6 log10 in cell cultures at 48-72 h post-infection, in a non-toxic and dose-responsive manner. CONCLUSIONS: The data indicate that PPMO have the ability to potently and specifically suppress SARS-CoV-2 growth and are promising candidates for further preclinical development.

The ETS transcription factor ELF1 regulates a broadly antiviral program distinct from the type I interferon response.

Induction of vast transcriptional programs is a central event of innate host responses to viral infections. Here we report a transcriptional program with potent antiviral activity, driven by E74-like ETS transcription factor 1 (ELF1). Using microscopy to quantify viral infection over time, we found that ELF1 inhibits eight diverse RNA and DNA viruses after multi-cycle replication. Elf1 deficiency results in enhanced susceptibility to influenza A virus infections in mice. ELF1 does not feed-forward to induce interferons, and ELF1's antiviral effect is not abolished by the absence of STAT1 or by inhibition of JAK phosphorylation. Accordingly, comparative expression analyses by RNA-seq revealed that the ELF1 transcriptional program is distinct from interferon signatures. Thus, ELF1 provides an additional layer of the innate host response, independent from the action of type I interferons.

Respiratory responses to external ammonia in zebrafish (Danio rerio).

The effects of high external ammonia (HEA) exposure on breathing and the potential involvement of ammonia transporting Rh proteins in ammonia sensing were assessed in larval and adult zebrafish. Acute exposure of adults to either 250 or 500 µM (NH4)2SO4 caused increases in ventilation amplitude (AVENT) without affecting frequency (fVENT), resembling the ventilatory response to hypercapnia rather than hypoxia, during which fVENT was increased exclusively. The hyperventilatory response to HEA was prevented by hyperoxia, indicating that control of breathing through ammonia sensing is likely secondary to O2 chemoreception. Neuroepithelial cells (NECs) isolated from gill filaments exhibited a significant increase of intracellular [Ca2+] in response to 1 mM NH4Cl but this response was small (roughly 30%) compared to the response to hypercapnia (37.5 mmHg; ~800% increase). Immunohistochemistry (IHC) failed to reveal the presence of Rh proteins (Rhcgb, Rhbg or Rhag) in gill filament NECs. Knockout of rhcgb did not affect the ventilatory response of adults to HEA. Larvae at 4 days post fertilization (dpf) responded to HEA with increases in fVENT (AVENT was not measured). The hyperventilatory response of larvae to HEA was attenuated (60% reduction) after treatment from 0 to 4 dpf with the sympathetic neurotoxin 6-hydroxydopamine. In larvae, Rhcgb, Rhbg and Rhag were undetectable by IHC in cutaneous NECs yet the fVENT to HEA following Rhbg knockdown was slightly (22%) attenuated. Thus, the hyperventilatory response to external ammonia in adult zebrafish, while apparently initiated by activation of NECs, does not require Rhcgb, nor is the entry of ammonia into NECs reliant on other Rh proteins. The lack of colocalization of Rh proteins with NECs suggests that the entry of ammonia into NECs in larvae, also is not facilitated by this family of ammonia channels.

Comprehensive analysis of fatty acids in human milk of four Asian countries.

Human milk lipids provide not only energy but also indispensable bioactive components such as essential fatty acids. To establish the recommended daily intake value and guidelines for infant formula, a reference library of fatty acid composition has been generated from 4 Asian countries (South Korea, China, Vietnam, and Pakistan). Regardless of country, palmitic acid (C16:0), linoleic acid (C18:1), and linolenic acid (C18:2) were the 3 most abundant fatty acids in human milk and account for more than 75% of total fatty acids (total FA). However, there were several considerable differences between fatty acids, particularly n-3 and n-6 (omega-3 and omega-6) groups. Chinese mothers' milk had a high concentration of linoleic acid at 24.38 ± 10.02% of total FA, which may be due to maternal diet. Among the 4 countries, Pakistani mothers' milk contained a high amount of saturated fatty acid (56.83 ± 5.96% of total FA), and consequently, polyunsaturated fatty acids, including n-3 and n-6, were significantly lower than in other countries. It is noteworthy that docosahexaenoic acid (DHA) in Pakistani mothers' milk was 44.8 ± 33.3 mg/L, which is only 25 to 30% of the levels in the other 3 countries, suggesting the need for DHA supplementation for infants in Pakistan. Moreover, the ratio of n-6 to n-3 was also remarkably high in Pakistani mothers' milk (15.21 ± 4.96), being 1.4- to 1.7-fold higher than in other countries. The average DHA:ARA ratio in Asian human milk was 1.01 ± 0.79. Korean mothers' milk showed a high DHA:ARA ratio, with a value of 1.30 ± 0.98, but Pakistani mothers' milk had a significantly lower value (0.42 ± 0.12). The fatty acid compositions and anthropometric data of mother (body mass index, age) did not show any correlation. The obtained data might provide information about human milk compositions in the Asian region that could benefit from setting up recommended nutrient intake and infant formula for Asian babies.

A Dystrophin Exon-52 Deleted Miniature Pig Model of Duchenne Muscular Dystrophy and Evaluation of Exon Skipping.

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by mutations in the DMD gene and the subsequent lack of dystrophin protein. Recently, phosphorodiamidate morpholino oligomer (PMO)-antisense oligonucleotides (ASOs) targeting exon 51 or 53 to reestablish the DMD reading frame have received regulatory approval as commercially available drugs. However, their applicability and efficacy remain limited to particular patients. Large animal models and exon skipping evaluation are essential to facilitate ASO development together with a deeper understanding of dystrophinopathies. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, we generated a Yucatan miniature pig model of DMD with an exon 52 deletion mutation equivalent to one of the most common mutations seen in patients. Exon 52-deleted mRNA expression and dystrophin deficiency were confirmed in the skeletal and cardiac muscles of DMD pigs. Accordingly, dystrophin-associated proteins failed to be recruited to the sarcolemma. The DMD pigs manifested early disease onset with severe bodywide skeletal muscle degeneration and with poor growth accompanied by a physical abnormality, but with no obvious cardiac phenotype. We also demonstrated that in primary DMD pig skeletal muscle cells, the genetically engineered exon-52 deleted pig DMD gene enables the evaluation of exon 51 or 53 skipping with PMO and its advanced technology, peptide-conjugated PMO. The results show that the DMD pigs developed here can be an appropriate large animal model for evaluating in vivo exon skipping efficacy.

[Evaluation and application of two methads for the determination of red blood cells in urine].

Objective: This study was aimed to evaluate the accuracy and conformity of the dry chemical urine analyzer UA-5800 and the urine formed element analyzer EH-2080Cused in the detection of urine red blood cells, and establish screening and retesting standards for urine red blood cell testing. Methods: Automatic urine analysis line EU-8000 (composed of dry chemical urine analyzer UA-5800 and urine formed element analyzer EH-2080C) was used to conduct hematuria and urine erythrocyte test in 2336 fresh urine samples from occupational medical examinations in our hospital, the sensitivity (true positive rate) , specificity (true negative rate) and the positive coincidence rate (positive rate) and negative coincidence rate (negative rate) of the corresponding test results were analyzed tow instruments. Results: The sensitivity of urinary occult blood detected by UA-5800 and urinary erythrocytes examined by EH-2080C were 98.33% and 95.00%, there was no statistical significant between the two results (χ(2)=0.50、P>0.05) ; specificity was 82.47% and 97.45% respectively, which existed statistical significant (χ(2)=337.05、P<0.01) . Comparing the two methods, the statistical significant remained (χ(2)=339.05、P<0.01) . Among all results of urinary occult blood detected, the EH-2080C had the highest urinary erythrocyte negative coincidence rate (99.89%) ; while in the comparison of urinary occult blood positive grades at 3+, the highest positive coincidence rate of urinary erythrocyte in EH-2080C was 85.76%; when the urine occult blood levels were weakly positive, 1+, 2+, the positive coincidence rate of EH-2080C examination was low (8.97%, 14.40% and 36.48%) , which increased with elevated positive grade. Conclusion: The positive specimens screened by automatic urine analysis line were examined by gold standard microscope combining with with the criteria of urine red blood cell screening and retesting in our laboratory, which can ensure high accuracy and high efficiency of urine erythrocyte analysis.

TMPRSS2 Is the Major Activating Protease of Influenza A Virus in Primary Human Airway Cells and Influenza B Virus in Human Type II Pneumocytes.

Cleavage of influenza virus hemagglutinin (HA) by host cell proteases is essential for virus infectivity and spread. We previously demonstrated in vitro that the transmembrane protease TMPRSS2 cleaves influenza A virus (IAV) and influenza B virus (IBV) HA possessing a monobasic cleavage site. Subsequent studies revealed that TMPRSS2 is crucial for the activation and pathogenesis of H1N1pdm and H7N9 IAV in mice. In contrast, activation of H3N2 IAV and IBV was found to be independent of TMPRSS2 expression and supported by an as-yet-undetermined protease(s). Here, we investigated the role of TMPRSS2 in proteolytic activation of IAV and IBV in three human airway cell culture systems: primary human bronchial epithelial cells (HBEC), primary type II alveolar epithelial cells (AECII), and Calu-3 cells. Knockdown of TMPRSS2 expression was performed using a previously described antisense peptide-conjugated phosphorodiamidate morpholino oligomer, T-ex5, that interferes with splicing of TMPRSS2 pre-mRNA, resulting in the expression of enzymatically inactive TMPRSS2. T-ex5 treatment produced efficient knockdown of active TMPRSS2 in all three airway cell culture models and prevented proteolytic activation and multiplication of H7N9 IAV in Calu-3 cells and H1N1pdm, H7N9, and H3N2 IAV in HBEC and AECII. T-ex5 treatment also inhibited the activation and spread of IBV in AECII but did not affect IBV activation in HBEC and Calu-3 cells. This study identifies TMPRSS2 as the major HA-activating protease of IAV in human airway cells and IBV in type II pneumocytes and as a potential target for the development of novel drugs to treat influenza infections.IMPORTANCE Influenza A viruses (IAV) and influenza B viruses (IBV) cause significant morbidity and mortality during seasonal outbreaks. Cleavage of the viral surface glycoprotein hemagglutinin (HA) by host proteases is a prerequisite for membrane fusion and essential for virus infectivity. Inhibition of relevant proteases provides a promising therapeutic approach that may avoid the development of drug resistance. HA of most influenza viruses is cleaved at a monobasic cleavage site, and a number of proteases have been shown to cleave HA in vitro This study demonstrates that the transmembrane protease TMPRSS2 is the major HA-activating protease of IAV in primary human bronchial cells and of both IAV and IBV in primary human type II pneumocytes. It further reveals that human and murine airway cells can differ in their HA-cleaving protease repertoires. Our data will help drive the development of potent and selective protease inhibitors as novel drugs for influenza treatment.