UNC45A-related osteo-oto-hepato-enteric syndrome in a Chinese neonate.

Unexplained diarrhea and cholestasis are common clinical phenotypes in newborns, indicating there is only a little common genetic basis for these conditions. However, it has been reported that defects in the UNC45A gene can lead to osteo-oto-hepato-enteric syndrome. However, to date, only 10 patients with this syndrome have been reported in 2 studies; therefore, there is still a lack of analysis regarding the correlation between disease phenotype and genotype. Trio-whole exome sequencing was conducted using DNA samples from a newborn with congenital diarrhea and cholestasis from a Chinese Han family. The UNC45A variants were verified using Sanger sequencing. In addition, we applied a crystal structure model to analyze the potential hazards associated with the variants. The plasmids were constructed in vitro and transfected into human 293T cells for Western blot (WB) analysis. After the mutant protein was fused with the Green Fluorescent Protein label, intracellular localization was observed using laser confocal microscopy. The gene detection results showed that the UNC45A gene of the newborn examined in the present study harbored the compound heterozygous variants p.Arg819Ter, and p.Leu237Pro; this was confirmed via Sanger sequencing. Analysis of the Leu237Pro crystal structure model suggested that this variant may decrease local structural stability and affect protein function. The Western blot and laser confocal microscopy observation results suggested that the Leu237Pro mutation leads to reduced protein expression, while the Arg819Ter mutation completely inhibits the expression of the protein. The compound heterozygous variants of UNC45A (p.Arg819Ter and p.Leu237Pro) may be pathogenic factors of congenital diarrhea and cholestasis in this neonatal patient. Therefore, UNC45A deficiency should be considered when intractable diarrhea and cholestasis occur in newborns.

Serum neuron-specific enolase, magnetic resonance imaging, and electrophysiology for predicting neurodevelopmental outcomes of neonates with hypoxic-ischemic encephalopathy: a prospective study.

BACKGROUND: Neonatal hypoxic-ischemic encephalopathy (HIE) is an important cause of mortality and morbidity. Effective indicators for the early diagnosis of brain injury after HIE and prognosis are lacking. This study aimed to examine the predictive value of serum neuron-specific enolase (NSE), amplitude-integrated electroencephalography (aEEG), and magnetic resonance imaging (MRI), alone and in combination, for the neurological outcomes in neonates with HIE. METHODS: Newborns with HIE born and treated at the Third Affiliated Hospital of An-Hui Medical University were consecutively included in this prospective cohort study (June 2013 to December 2020). Encephalopathy was classified as mild, moderate or severe according to Samat and Sarnat. All patients were assessed serum 1-day NSE and 3-day NSE levels after birth. The children were classified by neurological examination and Bayley Scales of Infant Development II at 18 months of age. ROC analysis was used to evaluate the predictive accuracy of the neurodevelopment outcomes. RESULTS: A total of 50 HIE neonates were enrolled (normal group: 32 (64.0%), moderate delay: 5 (10.0%), severe delay: 30(26.0%)) according to Bayley II scores. Serum 3-day NSE levels increased with worsening neurodevelopment outcomes (normal: 20.52 ± 6.42 µg/L vs. moderate: 39.82 ± 5.92 µg/L vs. severe: 44.60 ± 9.01 µg/L, P < 0.001). The MRI findings at 4-7 days after birth were significantly different among the three groups (P < 0.001). Forty-two (84.0%) children had abnormal aEEG. The combination of the three abnormalities combined together had 100% sensitivity, 97.70% specificity, 98.25% PPV, and 99.98% NPV. CONCLUSIONS: MRI, aEEG, and 3-day NSE can predict the neurological prognosis of newborns with HIE without hypothermia treatment. Their combination can improve the predictive ability for long-term neurobehavioral prognosis.

Neonatal myofibrillar myopathy type II associated with biallelic UNC-45B gene novel mutation and perinatal myasthenia as the core phenotype: A case report.

Myofibrillar myopathy (MFM) is characterized by phenotypic heterogeneity; decreased function of the myosin-directed chaperone, UNC-45B protein, leads to MFM II, which is characterized by slow progressive proximal myasthenia. Currently, only two studies have reported 11 cases worldwide. This study aimed to conduct genetic research and etiological analysis of a neonatal case of perinatal myasthenia who eventually died due to autonomic dyspnea. The case involved a newborn female admitted for weak cries and groaning. Physical examination revealed shallow and irregular spontaneous breathing, difficulty feeding, hip flexion and knee flexion in both lower limbs, hypotonia (level 1), less translation action, and inability to resist gravity. The child died at 23 days after birth. Gene testing, mutation analysis, and crystal structure analysis were conducted. Cell culture and plasmid construction were conducted, followed by western blot analysis. Pathological changes, including Z-line breakage, were observed in the muscle biopsies of different tissues. Gene testing showed that UNC-45B had a novel compound heterozygous mutation (c.2357T>A/p.Met786Lys, c.2591A>C/p.His864Pro), and in vitro functional experiments showed that the variants could lead to a decrease in protein expression. This study expands the UNC-45B mutation and phenotype spectrum by reporting an MFM II case in a Chinese patient for the first time.

Liposome Encapsulation of Oncolytic Virus M1 To Reduce Immunogenicity and Immune Clearance in Vivo.

Oncolytic viral therapy is an attractive novel strategy for cancer therapy. As a natural alphavirus, oncolytic virus M1 is able to infect and kill various zinc finger antiviral protein (ZAP)-deficient tumor cells selectively, while leaving normal cells undamaged. However, M1 can trigger the production of neutralizing antibodies that dramatically weaken its antitumor effect. In order to attenuate immunogenicity of the therapeutic M1 virus, we encapsulated it into liposomes (referred to as M-LPO) using the thin-film hydration method. The effect of anti-M1 neutralizing antibody on M-LPO was examined in LoVo and Hep 3B cell lines. In the absence of neutralizing antibodies, treating cells with naked M1, blank liposomes (LPO), M-LPO, or a simple mixture of M1 and liposomes (LPO+M1) inhibited cell growth. In the presence of neutralizing antibodies, only M-LPO inhibited cell growth. After intravenous administration, M-LPO reduced the production of the M1-neutralizing antibody and the corresponding immune response. Analysis of the M-LPO uptake by cells was examined by confocal microscopy using M1 labeled with FITC and liposomal shells labeled with RhB. The results suggest that M1 may be released from liposomes before or after M-LPO internalization. Taken together, our results suggest that encapsulating oncolytic virus M1 in liposomes may reduce intrinsic viral immunogenicity for improved anticancer therapy.

[Association between maternal pre-pregnancy body mass index and adverse outcomes of late preterm infants].

OBJECTIVE: To study the association between maternal pre-pregnancy body mass index (BMI) and adverse outcomes of late preterm infants (LPI). METHODS: A total of 367 LPI who were born from January 2011 to December 2015 and admitted to the neonatal ward were enrolled. The BMI criteria for Chinese population were used to analyze the factors for maternal pre-pregnancy BMI and its association with adverse outcomes of LPI (1 minute Apgar score ≤7, delivery room resuscitation, hospitalization days after birth >7 days, and ventilation duration ≥6 hours). RESULTS: Of all LPIs, there were 64 LPI (17.4%) in the low maternal pre-pregnancy BMI group, 243 LPI (66.2%) in the normal maternal pre-pregnancy BMI group, and 60 LPI (16.4%) in the high maternal pre-pregnancy BMI group. Low pre-pregnancy BMI was the risk factor for 1 minute Apgar score ≤7 (OR=3.243, 95% CI: 1.102-9.546) and need for delivery room resuscitation (OR=3.492, 95%CI: 1.090-11.190), and high pre-pregnancy BMI was the risk factor for hospitalization days after birth >7 days (OR=1.992, 95%CI: 1.024-3.874). CONCLUSIONS: Abnormal maternal pre-pregnancy BMI has adverse effects on the outcomes of LPI. In order to reduce these adverse outcomes BMI should be controlled within the normal range in pregnant women.

Sex differences in brain MRI abnormalities and neurodevelopmental outcomes in a rat model of neonatal hypoxia-ischemia.

PURPOSE/AIM OF THE STUDY: Hypoxic-ischemic brain injury (HIBI) is associated with high mortality and neurodevelopmental deficits. We explored gender influence in a HIBI rat model. MATERIALS AND METHODS: Sprague-Dawley rats underwent HIBI on postnatal day (P) 7. Nervous reflexes, footprints, Morris water maze performances and magnetic resonance imaging (MRI) were analyzed. RESULTS: Mortality rate was higher in HIBI males (20%) than in females (12.5%). Growth rate was slower in the HIBI group (p < 0.05), but was similar between HIBI males and females. HIBI rats showed impaired performances in the cliff aversion reflex, negative geotaxis reflex and gait tests at P14 (p < 0.05), but not at P9 or P11. There were no significant differences for the cliff aversion reflex and gait tests between genders. Negative geotaxis reflex impairment at P14 was more severe in HIBI males (p < 0.05). Step length and toe distance contralateral (but not ipsilateral) to the cerebral damage were shorter in HIBI rats, and were shorter in HIBI males than females (p < 0.05). Morris water maze latency time and swimming distance were longer in the HI group than in controls, and prolonged in HIBI males compared with females (p < 0.05). In the HIBI group, MRI showed more severe injury at P10 and P67 in males than in females (p < 0.05). CONCLUSIONS: Male rats are more vulnerable to the detrimental consequences of HIBI, with more severe nervous reflex deficits, brain injury, memory impairment and hemiplegic paralysis than female rats. Serial neurobehavioral follow-up is still executed on the HIBI infants who is absent of detectable abnormalities in early children.

Development of a novel berberine-mediated mitochondria-targeting nano-platform for drug-resistant cancer therapy.

Recent studies have shown that targeting doxorubicin to mitochondria of tumor cells can bypass the multi-drug resistance problem and inhibit tumor growth. We previously discovered that the C-9th and C-13th position-alkylated berberine derivatives possess improved mitochondria-targeting activity compared to berberine. Therefore, we hypothesize that these alkylated berberine derivatives could be utilized as potential mitochondrial-targeting ligands by inserting the alkyl chain into the liposomal bilayer membrane during the preparation of liposomes. In this research, a berberine derivate (a 16-carbon aliphatic chain was introduced to the C-9th of berberine, 9-C16 berberine) was employed to prepare mitochondria-targeting doxorubicin-loaded folic acid-conjugated polyethylene glycol(PEGylated) liposomes (MT-FOL-PLS). The results of in vitro cytotoxicity and apoptosis-inducing studies revealed that MT-FOL-PLS showed the strongest cytotoxicity and apoptosis-inducing effects in drug resistant MCF-7/adr cells in comparison with free doxorubicin and regular liposomal doxorubicin. MT-FOL-PLS enhanced cellular uptake of doxorubicin up to 15-fold compared to free doxorubicin, and targeted doxorubicin to mitochondria. In vivo and ex vivo drug distribution studies showed that MT-FOL-PLS increased the drug distribution in tumor and the administration of MT-FOL-PLS to resistant MCF-7/adr cell mouse xenografts stopped tumor growth. Our results confirmed that alkylated berberines can be exploited as mitochondrial-targeting ligands to overcome cancer multi-drug resistance, further advancing the research on active targeting of liposome delivery systems in the treatment of resistant cancer.

Preparation and evaluation of lipid polymer nanoparticles for eradicating H. pylori biofilm and impairing antibacterial resistance in vitro.

The resistance of Helicobacter pylori to classical antimicrobial treatment has become increasingly common, whereupon biofilms are considered to play an important role in the resistance mechanism. Here 10.2% of amoxicillin (AMX) and a novel anti H. pylori adhesion material pectin sulfate (PECS) loaded lipid polymer nanoparticles (LPN) were prepared, with rhamnolipid and phospholipids as the outer mixed lipids layer (RHL-PC-LPN). The size of RHL-PC-LPN was around 200 nm, was negatively-charged, and showed sustained and complete drug release within 24h. In an in vitro study, H. pylori biofilm models were successfully established. RHL-PC-LPN, superior to PC-LPN (employing phospholipids only as the outer lipid layer), PECS+AMX (mixture of PECS and AMX) and AMX only, was proven to significantly eradicate H. pylori in the biofilm form. In accordance to our previous results, the RHL-PC-LPN group, together with the PC-LPN and PECS+AMX group, inhibited H. pylori from adhering to AGS cells. Investigating the underlying mechanisms contributing to the death of H. pylori caused by RHL-PC-LPN, we found that LPN could lower the antibiotic minimal inhibition concentration (MIC) to biofilm form from 125 µg/ml to 15.6 µg/ml. Furthermore, FITC-ConA labeled extracellular polymeric substances (EPS) were decreased in the RHL-PC-LPN group observed by a laser scanning confocal microscope. Therefore, we conclude that employing the mixed lipids of rhamnolipid and phospholipids as the outer layer of nanoparticles and PECS as the inner core produces a system capable of significantly disrupting H. pylori biofilm by eliminating the EPS as well as inhibiting the adherence and colonization of bacteria.

Optimized mixed oils remarkably reduce the amount of surfactants in microemulsions without affecting oral bioavailability of ibuprofen by simultaneously enlarging microemulsion areas and enhancing drug solubility.

The toxicity and irritation associated with high amounts of surfactants restrict the extensive utilization of microemulsions. To address these shortcomings, employing mixed oils to enlarge microemulsion areas therefore reducing surfactant contents is a promising strategy. However, what kinds of mixed oils are more efficient in enlarging microemulsion areas still remains unclear. In this research, we found that the chain length and degree of unsaturation of oils play a key role in enlarging microemulsion areas. The combination of moderate chain saturated oil caprylic/capric triglyceride (GTCC) with long chain unsaturated oil glycerol trioleate significantly increased the microemulsion areas. Solubility of ibuprofen in the mixed oils was unexpectedly and remarkably increased (almost 300mg/mL) compared with that (around 100mg/mL) of the single oil (GTCC), which also resulted in greatly increased solubility of ibuprofen in mixed oils-containing microemulsions. By optimizing the mixed oil formulation, the absolute amount of surfactant in drug-loaded microemulsions was reduced but increased drug oral bioavailability in rats was maintained. It could be concluded that the combined use of moderate chain oils and long chain unsaturated oils could not only acquire enlarged microemulsion areas but also enhanced drug solubility, therefore doubly reducing surfactant amount, which is extremely beneficial for developing safe microemulsions.

Determination of neuroprotective oxysterols in Calculus bovis, human gallstones, and traditional Chinese medicine preparations by liquid chromatography with mass spectrometry.

So far, the components responsible for the neuroprotective effects of Calculus bovis are unclear. Cholesterol, one of the major components in Calculus bovis, is easily oxidized into oxysterols, which possess direct or indirect neuroprotective effects proved by our and others' previous studies. Therefore, a liquid chromatography with mass spectrometry method coupled with ultrasonic extraction and solid-phase extraction was developed for the determination of neuroprotective oxysterols in Calculus bovis, human gallstones, and traditional Chinese medicine preparations. Chromatographic separation was achieved on a C18 column with isocratic elution at a flow rate of 1 mL/min. The established method showed good linearity (R(2) > 0.998), sensitivity with low limits of detection (0.06-0.39 µg/g), acceptable precisions (relative standard deviations ≤ 7.4%), stability (relative standard deviations ≤ 5.9%), and satisfactory accuracy (92.4-102.9%) for all analytes identified by different retention times, which could be applied for the determination of oxysterols. Five kinds of oxysterols proved to function as neuroprotectants were detected at different concentrations. Among them, 7ß-hydroxycholesterol and cholestane-3ß,5α,6ß-triol were rather abundant in the samples. It could be concluded that the potential neuroprotective components in Calculus bovis may be these oxysterols.

Synthesis of taurine-fluorescein conjugate and evaluation of its retina-targeted efficiency in vitro.

In this work, retinal penetration of fluorescein was achieved in vitro by covalent attachment of taurine to fluorescein, yielding the F-Tau conjugate. Nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HRMS) were used to confirm the successful synthesis of F-Tau. The cellular uptake of F-Tau in adult retinal pigment epithelial cells (ARPE-19) and human retinal microvascular endothelial cells (hRMECs) was visualized via confocal scanning microscopy. The results indicated an improvement of solubility and a reduction of logP of F-Tau compared with fluorescein. As compared with fluorescein, F-Tau showed little toxicity, and was retained longer by cells in uptake experiments. F-Tau also displayed higher transepithelial permeabilities than fluorescein in ARPE-19 and hRMECs monolayer cells (P<0.05). These results showed that taurine may be a useful ligand for targeting small-molecule hydrophobic pharmaceuticals into the retina.

[Human umbilical cord blood mononuclear cell transplantation promotes long-term neurobehavioral functional development of newborn SD rats with hypoxic ischemic brain injury].

OBJECTIVE: To explore the effect of human umbilical cord blood mononuclear cells (UCBMC) promoting nerve behavior function and brain tissue recovery of neonatal SD rat with hypoxic ischemic brain injury (HIBI). METHOD: A modified newborn rat model that had a combined hypoxic and ischemic brain injury as described by Rice-Vannucci was used, early nervous reflex, the Morris water maze and walking track analysis were used to evaluate nervous behavioral function, and brain MRI, HE staining to evaluate brain damage recovery. RESULT: Newborn rat Rice-Vannucci model showed significant brain atrophy, obvious hemiplegia of contralateral limbs,e.g right step length [(7.67 ± 0.46) cm vs. (8.22 ± 0.50) cm, F = 1.494] and toe distance [(0.93 ± 0.06) cm vs. (1.12 ± 0.55) cm, F = 0.186] were significantly reduced compared with left side, learning and memory ability was significantly impaired compared with normal control group (P < 0.01); Cliff aversion [(8.44 ± 2.38) s vs.(14.22 ± 5.07) s, t = 4.618] and negative geotaxis reflex time [(7.26 ± 2.00) s vs. (11.76 ± 3.73) s, t = 4.755] on postnatal 14 days of HIBI+ transplantation group were significantly reduced compared with HIBI+NaCl group (P < 0.01) ; the Morris water maze experiment showed escape latency [ (23.11 ± 6.64) s vs. (34.04 ± 12.95) s, t = 3.356] and swimming distance [ (9.12 ± 1.21) cm vs.(12.70 ± 1.53) cm, t = 17.095] of HIBI+transplantation group were significantly reduced compared with those of HIBI+NaCl group (P < 0.01) ; the residual brain volume on postnatal 10 d [ (75.37 ± 4.53)% vs. (67.17 ± 4.08)%, t = -6.017] and 67 d [ (69.05 ± 3.58)% vs.(60.83 ± 3.69)%, t = -7.148]of HIBI+ transplantation group were significantly larger than those of HIBI+NaCl group (P < 0.01); After human UCBMC transplantation, left cortical edema significantly reduced and nerve cell necrosis of HIBI+ transplantation group is not obvious compared with HIBI+NaCl group. CONCLUSION: Human UCBMC intraperitoneal transplantation significantly promoted recovery of injured brain cells and neurobehavioral function development.

Bcl-2-associated X protein is the main mediator of manumycin a-induced apoptosis in anaplastic thyroid cancer cells.

We previously demonstrated that the combination of paclitaxel and manumycin A, a farnesyltransferase inhibitor, enhanced apoptosis of anaplastic thyroid cancer (ATC) cells. However, the mechanism of the manumycin-induced apoptosis is not fully understood. In this study, we discovered that mitochondrial ultrastructure condensation occurred after treatment with manumycin or manumycin plus paclitaxel. Bongkrekic acid and cyclosporin A, which are known inhibitors of the voltage-dependent anion channel, failed to inhibit cytochrome c release induced by manumycin or manumycin plus paclitaxel, suggesting that mitochondrial permeability transition pores were not involved. We also found that manumycin induced translocation of Bcl-2-associated X protein (Bax), another possible mediator of cytochrome c release, from the cytosol to the mitochondria. Silencing Bax with a specific small interfering RNA blocked manumycin-induced mitochondrial condensation and cytochrome c release, arguing the dependence of manumycin-induced apoptosis on Bax. Using a binary adenoviral vector system, we found that overexpression of Bax enhanced manumycin-induced apoptosis of ATC cells, and the combination of manumycin and overexpression of Bax increased inhibition of ATC xenograft growth in nude mice. Thus, we concluded that manumycin-induced apoptosis in ATC cells was primarily mediated by Bax and that increasing Bax expression may sensitize ATC cells to manumycin.