Case report: Identification of a novel triplication of alpha-globin gene by the third-generation sequencing: pedigree analysis and genetic diagnosis.

BACKGROUND: Thalassemia, a common autosomal hereditary blood disorder worldwide, mainly contains α- and ß-thalassemia. The α-globin gene triplicates allele is harmless for carriers, but aggravates the phenotype of ß-thalassemia. Therefore, it is particularly crucial to accurately detect the structural variants of α-globin gene clusters. CASE REPORT: We reported a 28-year-old man, the proband, with microcytic hypochromic anemia. From pedigree analysis, his mother and sister had hypochromic microcytosis, and his father was normal. Genetic testing of thalassemia identified a novel α-globin gene triplicate named αααanti4.2del726bp (NC_000016.10:g.170769_174300dupinsAAAAAA) by third-generation sequencing (TGS) in the proband and his father, which was further validated by multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing. The genotypes of the proband's mother and sister were both -α3.7/αα compounded with heterozygous HBB:c.126_129delCTTT. They were categorized as silent α-thalassemia with co-inheritance of ß-thalassemia trait. The proband's genotype additionally had the α-globin gene triplicates compared with his mother and sister, which increased the imbalance between α/ß-globin, so the proband had more severe hematological parameters. The proband's wife was diagnosed as HBA2:c.427T > C heterozygosis, and his daughter had the novel α-globin gene triplicates compounded with HBA2:c.427T > C, therefore the girl might be asymptomatic. CONCLUSION: The identification of the novel α-globin gene triplicates provides more insight for the research of thalassemia variants and indicates that TGS has significant advantages on genetic testing of thalassemia for the reliability, accuracy and comprehensiveness.

Case report: A novel 10.8-kb deletion identified in the ß-globin gene through the long-read sequencing technology in a Chinese family with abnormal hemoglobin testing results.

Background: Thalassemia is a common inherited hemoglobin disorder caused by a deficiency of one or more globin subunits. Substitution variants and deletions in the HBB gene are the major causes of ß-thalassemia, of which large fragment deletions are rare and difficult to be detected by conventional polymerase chain reaction (PCR)-based methods. Case report: In this study, we reported a 26-year-old Han Chinese man, whose routine blood parameters were found to be abnormal. Hemoglobin testing was performed on the proband and his family members, of whom only the proband's mother had normal parameters. The comprehensive analysis of thalassemia alleles (CATSA, a long-read sequencing-based approach) was performed to identify the causative variants. We finally found a novel 10.8-kb deletion including the ß-globin (HBB) gene (Chr11:5216601-5227407, GRch38/hg38) of the proband and his father and brother, which were consistent with their hemoglobin testing results. The copy number and exact breakpoints of the deletion were confirmed by multiplex ligation-dependent probe amplification (MLPA) and gap-polymerase chain reaction (Gap-PCR) as well as Sanger sequencing, respectively. Conclusion: With this novel large deletion found in the HBB gene in China, we expand the genotype spectrum of ß-thalassemia and show the advantages of long-read sequencing (LRS) for comprehensive and precise detection of thalassemia variants.

CNTN1 Aggravates Neuroinflammation and Triggers Cognitive Deficits in Male Mice by Boosting Crosstalk between Microglia and Astrocytes.

A wealth of knowledge regarding glial cell-mediated neuroinflammation, which contributes to cognitive deficits in Alzheimer's disease (AD) has emerged in recent years. Contactin 1(CNTN1), a member of the cell adhesion molecule and immunoglobulin supergene family, is centrally involved in axonal growth regulation and is also a key player in inflammation-associated disorders. However, whether CNTN1 plays a role in inflammation-related cognitive deficits and how this process is triggered and orchestrated remain to be fully elucidated. In this study, we examined postmortem brains with AD. CNTN1 immunoreactivity was markedly increased, particularly in the CA3 subregion, as compared with non-AD brains. Furthermore, by applying an adeno-associated virus-based approach to overexpress CNTN1 directly via stereotactic injection in mice, we demonstrated that hippocampal CNTN1 overexpression triggered cognitive deficits detected by novel object-recognition, novel place-recognition and social cognition tests. The mechanisms underlying these cognitive deficits could be attributed to hippocampal microglia and astrocyte activation, which led to aberrant expression of excitatory amino acid transporters (EAAT)1/EAAT2. This resulted in long-term potentiation (LTP) impairment that could be reversed by minocyline, an antibiotic and the best-known inhibitor of microglial activation. Taken together, our results identified Cntn1 as a susceptibility factor involved in regulating cognitive deficits via functional actions in the hippocampus. This factor correlated with microglial activation and triggered astrocyte activation with abnormal EAAT1/EAAT2 expression and LTP impairment. Overall, these findings may significantly advance our understanding of the pathophysiological mechanisms underlying the risk of neuroinflammation related cognitive deficits.

Case report: Long-read sequencing identified a novel 14.9-kb deletion of the α-globin gene locus in a family with α-thalassemia in China.

Background: Thalassemia is a hereditary blood disease resulting from globin chain synthesis impairment because of α- and/or ß-globin gene variants. α-thalassemia is characterized by non-deletional and deletional variants in the HBA gene locus, of which rare deletional variants are difficult to detect by conventional polymerase chain reaction (PCR)-based methods. Case report: We report the case of a one-month-old boy, who and his mother had abnormal hematological parameters, while his father had normal hematology. Conventional PCR-reverse dot blot (RDB) was performed for all family members to analyze the 23 most common thalassemia variants in China, but did not identify any pathologic variants. Single-molecule real-time (SMRT) long-read sequencing (LRS) technology was then performed and identified an unreported 14.9-kb large deletion (hg38 chr16:168,803-183,737) of the α-globin gene locus, which disrupted both HBA1 and HBA2 genes in the proband and his mother. The exact breakpoints of the deletion were confirmed by gap-PCR and Sanger sequencing. Conclusion: We have detected a novel large deletion in α-globin gene locus in China, which not only enriches the variant spectrum of thalassemia, but also demonstrates the accuracy and efficiency of LRS in detecting rare and novel deletions.

Infralimbic YTHDF1 is necessary for the beneficial effects of acute mild exercise on auditory fear extinction retention.

Exposure therapy is the most effective approach of behavioral therapy for anxiety and post-traumatic stress disorder (PTSD). But fear is easy to reappear even after successful extinction. So, identifying novel strategies for augmenting exposure therapy is rather important. It was reported that exercise had beneficial effects on cognitive and memory deficits. However, whether exercise could affect fear memory, especially for fear extinction remained elusive. Here, our results showed that exposure to acute mild exercise 1 or 2 h before extinction training can augment recent fear extinction retention and 2 h for the remote fear extinction retention. These beneficial effects could be attributed to increased YTHDF1 expression in medial prefrontal cortex (mPFC). Furthermore, by using an AAV-shRNA-based approach to silence YTHDF1 expression via stereotactic injection in prelimbic cortex (PL) or infralimbic cortex (IL), respectively, we demonstrated that silence YTHDF1 in IL, but not in PL, blunted augmentation of exposure therapy induced by acute mild exercise and accompanied with decreased NR2B and GluR1 expression. Moreover, YTHDF1 modulated dendritic spines remodeling of pyramidal neuron in IL. Collectively, our findings suggested that acute mild exercise acted as an effective strategy in augmenting exposure therapy with possible implications for understanding new treatment underlying PTSD.

Extensive Perivillous Fibrin and Intervillous Histiocytosis in a SARS-CoV-2 Infected Placenta From an Uninfected Newborn: A Case Report Including Immunohistochemical Profiling.

Placental infection by SARS-CoV-2 with various pathologic alterations reported. Inflammatory findings, such as extensive perivillous fibrin deposition and intervillous histiocytosis, have been postulated as risk factors for fetal infection by SARS-CoV-2. We describe the placental findings in a case of a 31-year-old mother with SARS-CoV-2 infection who delivered a preterm female neonate who tested negative for SAR-CoV2 infection. Placental examination demonstrated a small for gestational age placenta with extensive intervillous histiocytosis, syncytiotrophoblast karyorrhexis, and diffuse intervillous fibrin deposition. Immunohistochemical staining demonstrated infection of the syncytiotrophoblasts by SARS-CoV-2 inversely related to the presence of intervillous histiocytes and fibrin deposition. Our case demonstrates that despite extensive placental pathology, no fetal transmission of SARS-CoV-2 occurred, as well as postulates a relationship between placental infection, inflammation, and fibrin deposition.

Expression of Cntn1 is regulated by stress and associated with anxiety and depression phenotypes.

In recent years, our understanding of neural circuits associated with depression has increased. Although inherited factors are known to influence individual differences in the risk for this disorder, it has been difficult to identify specific genes that moderate circuit functions affecting depression. Genome-wide association studies have identified genetic variants of Cntn1 that are linked to major depressive disorders. Cntn1, a subset of the neural cell adhesion protein and immunoglobulin supergene family, participates in cell contact formation and axonal growth control and plays a role in degenerative and inflammatory disorders. However, neuronal substrates that mediate Cntn1 action on depression-like phenotypes and involved mechanisms are unclear. Here, we exploited chronic unpredictable stress (CUS) exposure and found that CUS treatment significantly increased hippocampal Cntn1 messenger RNA and protein expression in both mice and rats, but not in the medial prefrontal cortex, which presented a region-specific regulation. Using an adeno-associated virus-based approach to directly overexpress Cntn1 via stereotactic injection, we demonstrated that Cntn1 overexpression in the hippocampus triggered anxiety- and depression-like phenotypes in addition to microglia activation or phagocytosis in the hippocampus, resulting in upregulation of pro-inflammatory cytokine (IL1α, IL6, and Ccl2) mRNA expression and downregulation of anti-inflammatory cytokine (IL4 and CD206) mRNA expression, determined using real-time quantitative PCR, thus impairing hippocampal immature neurons in the dentate gyrus, determined using immunohistochemical staining for doublecortin, a specific marker for immature neurons. Collectively, our results identified Cntn1 as a novel risk gene involved in regulating anxiety and depression via functional actions in the hippocampus that is correlated with microglial activation or phagocytosis and reduced hippocampal immature neurons. These results may provide a better understanding of the pathophysiological mechanisms underlying the risk of depression-related disorders.

Peroxisome proliferator-activated receptor γ coactivator-1α is a central negative regulator of vascular senescence.

OBJECTIVE: Cellular senescence influences organismal aging and increases predisposition to age-related diseases, in particular cardiovascular disease, a leading cause of death and disability worldwide. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is a master regulator of mitochondrial biogenesis and function, oxidative stress, and insulin resistance. Senescence is associated with telomere and mitochondrial dysfunction and oxidative stress, implying a potential causal role of PGC-1α in senescence pathogenesis. APPROACH AND RESULTS: We generated a PGC-1α(+/-)/apolipoprotein E(-/-) mouse model and showed that PGC-1α deficiency promotes a vascular senescence phenotype that is associated with increased oxidative stress, mitochondrial abnormalities, and reduced telomerase activity. PGC-1α disruption results in reduced expression of the longevity-related deacetylase sirtuin 1 (SIRT1) and the antioxidant catalase, and increased expression of the senescence marker p53 in aortas. Further, angiotensin II, a major hormonal inducer of vascular senescence, induces prolonged lysine acetylation of PGC-1α and releases the PGC-1α-FoxO1 complex from the SIRT1 promoter, thus reducing SIRT1 expression. The phosphorylation-defective mutant PGC-1α S570A is not acetylated, is constitutively active for forkhead box O1-dependent SIRT1 transcription, and prevents angiotensin II-induced senescence. Acetylation of PGC-1α by angiotensin II interrupts the PGC-1α-forkhead box O1-SIRT1 feed-forward signaling circuit leading to SIRT1 and catalase downregulation and vascular senescence. CONCLUSIONS: PGC-1α is a primary negative regulator of vascular senescence. Moreover, the central role of posttranslational modification of PGC-1α in regulating angiotensin II-induced vascular senescence may inform development of novel therapeutic strategies for mitigating age-associated diseases, such as atherosclerosis.

Mitsuaria chitosanase with unrevealed important amino acid residues: characterization and enhanced production in Pichia pastoris.

A chitosan plate assay was employed to screen for chitosanase-producing bacterial strains and isolate 141 was found to exhibit high activity. Characterization of this isolate revealed that it belonged to Mitsuaria (designated as Mitsuaria sp. 141). The encoded chitosanase (choA) gene was then cloned by PCR and the deduced amino acid sequence showed 98% identity to a formerly described Mitsuaria chitosanitabida 3001 ChoA (McChoA). Surprisingly, the ChoA encoded by Mitsuaria sp. 141 (MsChoA) appeared to have a much higher optimum temperature compared to McChoA. Site-directed mutagenesis was then employed to generate five MschoA mutant genes encoding MsChoA K204Q, R216K, T222N, R216K/T222N, or K204Q/R216K/T222N. All the ChoA mutants exhibited a much lower specific activity and a lower optimum temperature. The results confirmed that the substitution of three non-conserved amino acids accounts for the major reduction of the enzyme activity in MsChoA. Furthermore, the MschoA gene was cloned for over-expression in Pichia pastoris after coding sequence optimization. One of the P. pastoris transformants with Mut(S) phenotype was found to produce 1,480.2 ± 340.9 U ChoA mL(-1) of cell culture by high-cell-density fermentation. This represents the highest yield of recombinant ChoA production that has ever been reported thus far. The recombinant P. pastoris strain should therefore be well suited for industrial-scale production of chitosanase.

PGC-1 alpha serine 570 phosphorylation and GCN5-mediated acetylation by angiotensin II drive catalase down-regulation and vascular hypertrophy.

Angiotensin II (Ang II) is a pleuripotential hormone that is important in the pathophysiology of multiple conditions including aging, cardiovascular and renal diseases, and insulin resistance. Reactive oxygen species (ROS) are important mediators of Ang II-induced signaling generally and have a well defined role in vascular hypertrophy, which is inhibited by overexpression of catalase, inferring a specific role of H(2)O(2). The molecular mechanisms are understood incompletely. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha) is a key regulator of energy metabolism and ROS-scavenging enzymes including catalase. We show that Ang II stimulates Akt-dependent PGC-1 alpha serine 570 phosphorylation, which is required for the binding of the histone acetyltransferase GCN5 (general control nonderepressible 5) to PGC-1 alpha and for its lysine acetylation. These sequential post-translational modifications suppress PGC-1 alpha activity and prevent its binding to the catalase promoter through the forkhead box O1 transcription factor, thus decreasing catalase expression. We demonstrate that overexpression of the phosphorylation-defective mutant PGC-1 alpha (S570A) prevents Ang II-induced increases in H(2)O(2) levels and hypertrophy ([(3)H]leucine incorporation). Knockdown of PGC-1 alpha by small interfering RNA promotes basal and Ang II-stimulated ROS and hypertrophy, which is reversed by polyethylene glycol-conjugated catalase. Thus, endogenous PGC-1 alpha is a negative regulator of vascular hypertrophy by up-regulating catalase expression and thus reducing ROS levels. We provide novel mechanistic insights by which Ang II may mediate its ROS-dependent pathophysiologic effects on multiple cardiometabolic diseases.

Toward top-down determination of PEGylation site using MALDI in-source decay MS analysis.

A novel matrix assisted laser desorption/ionization (MALDI)-based mass spectrometric approach has been evaluated to rapidly analyze a custom designed PEGylated peptide that is 31 residues long and conjugated with 20 kDa linear polyethylene glycol (PEG) at the side chain of Lys. MALDI-TOF MS provided sufficiently high resolution to allow observation of each of the oligomers of the heterogeneous PEGylated peptide (m/Deltam of ca. 500), while a typical ESI-MS spectrum of this molecule was extremely complex and unresolved. Reflector in-source decay (reISD) analysis using MALDI-TOF MS was attempted to identify the PEGylation site at intact molecular level without any sample treatment. An reISD spectrum of the free peptide was observed with abundant c-, y-, and [z + 2]-fragment ion series, whereas, in the fragmented PEGylated peptide, the fragment ion series were truncated at the residue where PEG was attached. Therefore, a direct comparison of these top-down reISD spectra suggested the location of the PEGylation site. Results from this study demonstrate a clear analytical utility of the ISD technique to characterize structural aspects of heterogeneous biomolecules.

Potential of baboon endothelial progenitor cells for tissue engineered vascular grafts.

Thrombosis and intimal hyperplasia limit the usefulness of small caliber vascular grafts. While some improvements have been reported for grafts seeded with mature endothelial cells (EC), the harvesting of ECs from autologous sources, for example, veins or adipose tissue, remains problematic. More recently, endothelial progenitor cells (EPCs) have been considered a promising source of ECs because EPCs can be readily isolated from whole blood then rapidly expanded in vitro. Additionally, EPCs are increasingly recognized to play important roles in hemostasis, angiogenesis, and arterial injury repair. However, the characterization of EPCs in relevant animal models remains poorly defined. Accordingly, we have characterized the isolation, growth, and functional characteristics of Baboon EPCs (BaEPCs) to evaluate their potential for an autologous cell source for tissue engineered vascular grafts. BaEPCs were successfully cultured from the peripheral blood with an average population doubling time of 1.17 +/- 0.43 days. While the BaEPCs were positive for typical EC markers of vWF, CD31, VE-cadherin, VEGF-R2, Thrombomodulin, and E-selectin, there was reduced eNOS expression. The BaEPCs cell body and actin filaments align in the direction of flow typical of mature ECs. Thus while the lack of eNOS expression is worthy of investigation, EPCs are an attractive cell source for tissue engineered vascular grafts and the baboon model has great potential for continuing evaluations of these cells.

A SPME-GC procedure for monitoring peppermint flavor in tablets.

A method was developed using solid-phase microextraction (SPME) and gas chromatography to monitor the peppermint flavor loss in a taste-masked tablet formulation. This was accomplished by headspace sampling of two major components of peppermint: menthone and menthol. It was found that the excipients from the tablet produced an important matrix effect and that standard addition analysis was necessary for improved accuracy of the determination. The method was shown to be specific and precise. Furthermore, the method produced acceptable results with adequate quantitation limits to determine peppermint flavors in taste-masked tablets. The optimized extraction procedure was successfully used to monitor the stability of peppermint flavor in an oral solid formulation. The accelerated stability studies of the tablet showed that the menthone and menthol was lost in an exponential manner and levels off after several days of heat exposure.

Interaction of methylparaben preservative with selected sugars and sugar alcohols.

The interaction of methylparaben preservative with selected sugars (glucose, fructose, sucrose, lactose, maltose, cellobiose) and sugar alcohols (lactitol, maltitol) were demonstrated in this study. It was observed that the formation of transesterification reaction products between methylparaben and the selected sugars occurred only under mild reaction conditions (e.g., pH 7.4 at 50 degrees C ), which were confirmed by HPLC-UV studies and mass spectrometry. On the other hand, under alkaline conditions and high temperature, degradation of the sugars predominated. Because sugars could easily undergo many possible degradation reactions and isomerization including on-column anomerization, the chromatograms of the reaction products were more complicated than those obtained from sugar alcohols. Sucrose, a nonreducing sugar, was much more stable than other selected sugars. The chromatogram of the transesterification reaction products of methylparaben with sucrose clearly showed eight peaks, which were likely to correspond to the same number of hydroxyl groups of sucrose. To compare the rate of the transesterification reaction of methylparaben with sucrose to that with sorbitol, kinetic studies were carried out. Similar rate constants were observed: 5.4 x 10(-7) L mol(-1) s(-1) and 4.9 x 10(-7) L mol(-1) s(-1) for sucrose and sorbitol, respectively.

HPLC and LC-MS studies of the transesterification reaction of methylparaben with twelve 3- to 6-carbon sugar alcohols and propylene glycol and the isomerization of the reaction products by acyl migration.

Sugar alcohols and parabens are commonly used ingredients in oral suspension formulations. However, their possible incompatibility because of transesterification reaction is a concern during formulation development. In order to gain more knowledge about the reaction, a high-performance liquid chromatographic (HPLC) method is developed to separate the transesterification reaction products of methylparaben preservative with twelve 3- to 6-carbon sugar alcohols and propylene glycol. It is found that the number of peaks separated or partially separated correlate well with the number of distinct hydroxyl groups present in the sugar alcohol molecules. This means that all the hydroxyl groups in a sugar alcohol molecule can react with methylparaben to form transesterification reaction products. These products are positional isomers that have identical UV spectra with a maximum at 255 nm and the same m/z ratio for molecular ions by liquid chromatography-mass spectrometry. When isolated individually, they can isomerize (interconvert) under suitable conditions to form other positional isomers by intramolecular acyl migration. The acyl migration pathway for each of the isolated positional isomers from the transesterification reaction of methylparaben with sorbitol, ribitol, and xylitol is followed by HPLC. Based on the information, a tentative assignment of the six isomer peaks generated from the transesterification reaction between methylparaben and sorbitol is proposed.

Retinal melatonin production: role of proteasomal proteolysis in circadian and photic control of arylalkylamine N-acetyltransferase.

PURPOSE: Dynamic day-night changes in melatonin synthesis are regulated by changes in the activity of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase [AA-NAT]). Similarly, a light-induced decrease in AA-NAT activity at night rapidly suppresses melatonin synthesis. The purpose of the current study was to test the hypothesis that in vivo changes of AA-NAT activity in chicken retina homogenates parallel changes in AA-NAT protein. This led to examination of the role of proteasomal proteolysis in the regulation of retinal AA-NAT activity and protein levels. METHODS: Chickens, entrained to a 12-hour light-12-hour dark cycle, were assessed under various lighting conditions, in some cases after in vivo intravitreal administration of the protein synthesis inhibitor cycloheximide or lactacystin, an inhibitor of the 20S proteasome. Tissue homogenates were prepared, AA-NAT enzyme activity was measured, and immunoreactive protein was estimated by Western blot using an anti-chicken AA-NAT(1-21) serum. RESULTS: The abundance of AA-NAT protein in both the retina and pineal gland exhibited a daily rhythm that was statistically indistinguishable from that of AA-NAT's activity measured in tissue homogenates. Acute exposure to light at night rapidly decreased AA-NAT protein and activity in a parallel fashion. Administration of cycloheximide at night decreased retinal AA-NAT activity in darkness and enhanced the effect of light. The light-evoked suppression of retinal AA-NAT protein and activity was blocked by intravitreal injection of lactacystin, which also was found to increase AA-NAT activity, either at night or during the daytime. CONCLUSIONS: AA-NAT activity measured in tissue homogenates reflects the steady state level of enzyme protein. AA-NAT protein in the retina turns over rapidly, reflecting a balance of de novo synthesis and proteasomal proteolysis. The suppressive effects of light at night are due primarily to enhanced AA-NAT proteolysis.

Intraperitoneal infusion of homocysteine increases intimal hyperplasia in balloon-injured rat carotid arteries.

Hyperhomocysteinemia is a significant risk factor in atherosclerosis and thrombosis. However, its role in the development of intimal hyperplasia after arterial reconstructive procedures remains uncertain. We therefore studied the effect of homocysteine on intimal hyperplasia in a rat model of carotid artery balloon injury. Twenty-four Sprague-Dawley rats were divided into three groups: control (saline infusion), and low dose (0.14 mg/day) and high dose (0.71 mg/day) homocysteine delivered continuously via osmotic pumps implanted intraperitoneally. All animals underwent left common carotid artery balloon denudation with sacrifice after 14 days. Plasma homocysteine levels, intimal hyperplasia, and cell proliferation of rat carotid arteries were determined. In vitro rat smooth muscle cell (SMC) proliferation with homocysteine treatment was also performed. Plasma homocysteine levels at sacrifice were 1.80+/-0.35, 2.65+/-0.05 and 3.50+/-0.22 microM in three groups, respectively. Intimal hyperplasia developed in all balloon-injured arteries in both control and homocysteine-treated animals. The intimal area and intima/media area ratio were increased by 92% (P<0.05) and 105% (P<0.05), respectively, in the high dose-homocysteine-treated animals as compared to the control animals. Homocysteine (high dose) also significantly promoted the intimal cell proliferation (bromodeoxyuridine incorporation) by 2.2-fold as compared to controls. Furthermore, homocysteine treatment in the cell culture study showed a concentration-dependent increase of rat SMC proliferation. These data demonstrate that the continuous intraperitoneal administration of homocysteine significantly increases intimal hyperplasia and SMC proliferation after carotid artery balloon injury in the rat as well as in vitro SMC proliferation. This study suggests that, following arterial reconstructive procedures, elevated plasma homocysteine may increase the complications of clinical restenoses that are associated with intimal hyperplasia.