RNA Interference against ATP as a Gene Therapy Approach for Prostate Cancer.

Chemotherapeutic agents targeting energy metabolism have not achieved satisfactory results in different types of tumors. Herein, we developed an RNA interference (RNAi) method against adenosine triphosphate (ATP) by constructing an interfering plasmid-expressing ATP-binding RNA aptamer, which notably inhibited the growth of prostate cancer cells through diminishing the availability of cytoplasmic ATP and impairing the homeostasis of energy metabolism, and both glycolysis and oxidative phosphorylation were suppressed after RNAi treatment. Further identifying the mechanism underlying the effects of ATP aptamer, we surprisingly found that it markedly reduced the activity of membrane ionic channels and membrane potential which led to the dysfunction of mitochondria, such as the decrease of mitochondrial number, reduction in the respiration rate, and decline of mitochondrial membrane potential and ATP production. Meanwhile, the shortage of ATP impeded the formation of lamellipodia that are essential for the movement of cells, consequently resulting in a significant reduction of cell migration. Both the downregulation of the phosphorylation of AMP-activated protein kinase (AMPK) and endoplasmic reticulum kinase (ERK) and diminishing of lamellipodium formation led to cell apoptosis as well as the inhibition of angiogenesis and invasion. In conclusion, as the first RNAi modality targeting the blocking of ATP consumption, the present method can disturb the respiratory chain and ATP pool, which provides a novel regime for tumor therapies..

Isolation of full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of Escherichia coli.

Here we describe a facile and robust genetic selection for isolating full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of redox-engineered Escherichia coli cells. The method is based on the transport of a bifunctional substrate comprised of an antigen fused to chloramphenicol acetyltransferase, which allows positive selection of bacterial cells co-expressing cytoplasmic IgGs called cyclonals that specifically capture the chimeric antigen and sequester the antibiotic resistance marker in the cytoplasm. The utility of this approach is first demonstrated by isolating affinity-matured cyclonal variants that specifically bind their cognate antigen, the leucine zipper domain of a yeast transcriptional activator, with subnanomolar affinities, which represent a ~20-fold improvement over the parental IgG. We then use the genetic assay to discover antigen-specific cyclonals from a naïve human antibody repertoire, leading to the identification of lead IgG candidates with affinity and specificity for an influenza hemagglutinin-derived peptide antigen.

Superresolution live-cell imaging reveals that the localization of TMEM106B to filopodia in oligodendrocytes is compromised by the hypomyelination-related D252N mutation.

Hypomyelination leukodystrophies constitute a group of heritable white matter disorders exhibiting defective myelin development. Initially identified as a lysosomal protein, the TMEM106B D252N mutant has recently been associated with hypomyelination. However, how lysosomal TMEM106B facilitates myelination and how the D252N mutation disrupts that process are poorly understood. We used superresolution Hessian structured illumination microscopy (Hessian-SIM) and spinning disc-confocal structured illumination microscopy (SD-SIM) to find that the wild-type TMEM106B protein is targeted to the plasma membrane, filopodia, and lysosomes in human oligodendrocytes. The D252N mutation reduces the size of lysosomes in oligodendrocytes and compromises lysosome changes upon starvation stress. Most importantly, we detected reductions in the length and number of filopodia in cells expressing the D252N mutant. PLP1 is the most abundant myelin protein that almost entirely colocalizes with TMEM106B, and coexpressing PLP1 with the D252N mutant readily rescues the lysosome and filopodia phenotypes of cells. Therefore, interactions between TMEM106B and PLP1 on the plasma membrane are essential for filopodia formation and myelination in oligodendrocytes, which may be sustained by the delivery of these proteins from lysosomes via exocytosis.

The journey of a lifetime - development of Pfizer's COVID-19 vaccine.

It would be apt to say that one of the greatest accomplishments in modern medicine has been the development of vaccines against COVID-19, which had paralyzed the entire world for more than a year. Pfizer and BioNTech codeveloped the first COVID-19 vaccine that was granted emergency-use authorization or conditional approval in several regions globally. This article is an attempt to go 'behind-the-scenes' of this development process and highlight key factors that allowed us to move with this unprecedented speed, while adhering to normal vaccine-development requirements to generate the information the regulatory authorities needed to assess the safety and effectiveness of a vaccine to prevent an infectious disease, including quality and manufacturing standards. This is also a story of how Pfizer and BioNTech leveraged our combined skill sets and experience to respond to the global health crisis to progress this program swiftly while ensuring the compliance with our high-quality standards and keeping patient safety at the forefront. We will also highlight multiple other factors that were instrumental in our success.

Novel Insight into the Potential Pathogenicity of Mitochondrial Dysfunction Resulting from PLP1 Duplication Mutations in Patients with Pelizaeus-Merzbacher Disease.

Among the hypomyelinating leukodystrophies, Pelizaeus-Merzbacher disease (PMD) is a representative disorder. The disease is caused by different types of PLP1 mutations, among which PLP1 duplication accounts for ∼70% of the mutations. Previous studies have shown that PLP1 duplications lead to PLP1 retention in the endoplasmic reticulum (ER); in parallel, recent studies have demonstrated that PLP1 duplication can also lead to mitochondrial dysfunction. As such, the respective roles and interactions of the ER and mitochondria in the pathogenesis of PLP1 duplication are not clear. In both PLP1 patients' and healthy fibroblasts, we measured mitochondrial respiration with a Seahorse XF Extracellular Analyzer and examined the interactions between the ER and mitochondria with super-resolution microscopy (spinning-disc pinhole-based structured illumination microscopy, SD-SIM). For the first time, we demonstrated that PLP1 duplication mutants had closer ER-mitochondrion interfaces mediated through structural and morphological changes in both the ER and mitochondria-associated membranes (MAMs). These changes in both the ER and mitochondria then led to mitochondrial dysfunction, as reported previously. This work highlights the roles of MAMs in bridging PLP1 expression in the ER and pathogenic dysfunction in mitochondria, providing novel insight into the pathogenicity of mitochondrial dysfunction resulting from PLP1 duplication. These findings suggest that interactions between the ER and mitochondria may underlie pathogenic mechanisms of hypomyelinating leukodystrophies diseases at the organelle level.

Shotgun scanning glycomutagenesis: A simple and efficient strategy for constructing and characterizing neoglycoproteins.

As a common protein modification, asparagine-linked (N-linked) glycosylation has the capacity to greatly influence the biological and biophysical properties of proteins. However, the routine use of glycosylation as a strategy for engineering proteins with advantageous properties is limited by our inability to construct and screen large collections of glycoproteins for cataloguing the consequences of glycan installation. To address this challenge, we describe a combinatorial strategy termed shotgun scanning glycomutagenesis in which DNA libraries encoding all possible glycosylation site variants of a given protein are constructed and subsequently expressed in glycosylation-competent bacteria, thereby enabling rapid determination of glycosylatable sites in the protein. The resulting neoglycoproteins can be readily subjected to available high-throughput assays, making it possible to systematically investigate the structural and functional consequences of glycan conjugation along a protein backbone. The utility of this approach was demonstrated with three different acceptor proteins, namely bacterial immunity protein Im7, bovine pancreatic ribonuclease A, and human anti-HER2 single-chain Fv antibody, all of which were found to tolerate N-glycan attachment at a large number of positions and with relatively high efficiency. The stability and activity of many glycovariants was measurably altered by N-linked glycans in a manner that critically depended on the precise location of the modification. Structural models suggested that affinity was improved by creating novel interfacial contacts with a glycan at the periphery of a protein-protein interface. Importantly, we anticipate that our glycomutagenesis workflow should provide access to unexplored regions of glycoprotein structural space and to custom-made neoglycoproteins with desirable properties.

A novel nomogram for predicting the risk of epilepsy occurrence after operative in gliomas patients without preoperative epilepsy history.

OBJECTIVE: Epilepsy is a common complication in glioma patients after undergoing brain tumor surgery combined with chemotherapy and/or radiotherapy. Whether antiepileptic drug prophylaxis could be used in these patients remains an open question. The purpose of this study was to produce a model for predicting the risk of epilepsy occurrence in such patients. METHODS: The clinicopathologic data of glioma patients after tumor treatment were reviewed in this study. Univariate and multivariate logistic regression analyses were carried out to analyze the correlation between the clinicopathologic data and the risk of epilepsy occurrence. A nomogram was built according to the multivariate logistic regression model results. RESULTS: A total of 219 patients with gliomas were reviewed. Univariate analyses revealed that age, WHO glioma classification, CD34, EGFR, Ki67, MGMT, P53 and VIM were significantly associated with the risk of epilepsy occurrence. Multivariate analyses revealed that age, WHO glioma classification, CD34, EGFR, MGMT, and VIM were predictors of risk of epilepsy occurrence. A nomogram of the risk of epilepsy occurrence was built based on statistically significant variables from the multivariate logistic regression analysis. The c-index of the nomogram was 0.755 (95 % confidence interval (CI), 0.742-0.769). SIGNIFICANCE: This nomogram model provides reliable information about the risk of epilepsy occurrence for oncologists and neurological physicians.

Combination of structure-performance and shape-performance relationships for better biphasic release in electrospun Janus fibers.

In nature, the combination of composition, structure, and shape determines the matter's functional performance to a large extent. Inspired by which, two electrospun Janus nanofiber formulations were created using side-by-side electrospinning in this work. Tamoxifen citrate (TAM) was used as a model drug and ethyl cellulose (EC) and polyvinylpyrrolidone K60 (PVP) as the polymer carrier matrices. The fibers have linear cylindrical morphologies and distinct Janus structures by scanning electron microscopy. One side of the fibers took a round shape, while the other was crescent-shaped. The drug was present in both polymer matrices in the form of amorphous solid dispersions, owing to strong intermolecular interactions between drug and polymer. In vitro dissolution tests demonstrated that both sets of fibers could provide biphasic drug release due to the difference in solubility of PVP and EC. The different shape of TAM-EC and TAM-PVP side of the Janus structure resulted in a considerable variation in the drug release profiles. The Janus structure with crescent TAM-PVP side and round TAM-EC side gave a more rapid burst release in the first phase of release, and slower sustained release in the second phase. This work thus reports a new strategy for systematically developing advanced functional nanomaterials based on both shape- and structure-performance relationships.

Clinical Features, Risk Factors, and Early Prognosis for Wallerian Degeneration in the Descending Pyramidal Tract after Acute Cerebral Infarction.

BACKGROUND: Wallerian degeneration(WD) occurs in the descending pyramidal tract(DPT) after cerebral infarction commonly, but studies of its degree evaluation, influencing factors and effects on nervous function are still limited. OBJECTIVES: The purpose of this study was to describe these findings and estimate their clinical significance. METHODS: In total, 133 patients confirmed acute cerebral infarction and restricted diffusion in the DPT of the cerebral peduncle by MRI scans. These cases were retrospectively reviewed. We describe their clinical characteristics and analyze influence factors of WD, including the timespan from symptom onset to MRI and TOAST classification. Their NIHSS scores at admission and first 7 days NIHSS improvement rate after admission were also analyzed. RESULTS: These patients were divided into three groups by timespan ≤7 days(n = 45),7-14 days(n = 70) and >14 days(n = 18). The mean WD degree (%)of these three groups was 44.41 ± 22.51,52.35 ± 22.61and 44.31 ± 19.35,respectively(p = 0.122).According to the TOAST classification, the mean WD degree(%) of the cardioembolism group(n = 28, 62.80 ± 25.12) was significantly different from both the large-artery atherosclerosis group(n = 73,45.08 ± 20.03,p = 0.000) and the small-vessel occlusion group(n = 23,39.68 ± 16.95,p = 0.000). The mean NIHSS score upon admission of the WD degree≤50% group(n = 82,8.17 ± 5.87) was different from that of the >50% group(n = 51,11.31 ± 7.00)(p = 0.006). However, the mean 7 days NIHSS improvement rate(%) of the WD degree≤50% group(n = 79,11.83 ± 23.76)and >50% group(n = 50,13.40 ± 27.88) was not significantly different(p = 0.733). CONCLUSIONS: Early WD in ischemic stroke patients has a correlation with serious baseline functional defects. Therefore, we should give close attention to imaging change, especially in those with cardioembolism .

Suppressing the Phase Segregation with Potassium for Highly Efficient and Photostable Inverted Wide-Band Gap Halide Perovskite Solar Cells.

Wide-band gap (WBG) mixed-halide perovskites have drawn much attention because of their excellent optoelectronic properties and the potential to be deployed in tandem solar cells. Nevertheless, the bromine incorporation inevitably leads to photoinduced phase segregation in WBG mixed-halide perovskites. Herein, potassium is used to effectively suppress photoinduced phase segregation, which is visualized with confocal photoluminescence microscopy imaging. Strikingly, the potassium passivation not only inhibits the formation of the narrow-band gap subphase but also enhances the crystallinity of the WBG mixed-halide perovskite. In addition, the potassium-passivated WBG perovskite exhibits lower defect density, longer charge carrier lifetime, and better photostability. As a result, the optimized KI (2 mol %)-passivated WBG perovskite solar cells (PSCs) deliver a champion power conversion efficiency of 18.3% with negligible hysteresis. They maintain 98% of their initial efficiency after 400 h under 100 mW·cm-2 white light illumination in nitrogen.

A nanofiber-based drug depot with high drug loading for sustained release.

Nanostructures and their related structure-performance relationships for "efficacious, safe and convenient" drug delivery are playing a more and more important role in the fast development of nanopharmaceutics. In this study, a core-shell fiber based nano depot (ND) is prepared for achieving a high drug loading and meanwhile ensuring a zero-order drug sustained release profile. With cellulose acetate (CA) as a filament-forming polymeric matrix and ferulic acid (FA) as a model drug, a triaxial electrospinning was implemented to generate the ND. An elaborate strategy was exploited to ensure a continuous, robust and effective preparation. The strategy comprised a solvent mixture as the outer fluid, a mixed solution containing FA and CA with a high CA concentration as the spinnable middle fluid, and a pure drug solution as the inner liquid for loading enough FA in the NDs as much as possible. TEM and SEM demonstrated the core-shell structure of NDs. The NDs had a drug loading of 71.5 ± 4.6%. The in vitro dissolution tests demonstrated that the loaded FA was able to release through a zero-order kinetics of Q (FA released percentage) to t (release time): Q = 12.03 + 1.89 t (R = 0.9928) during the 48 h. Only 3.6% of the loading FA was released during the late tailing-off period. Three different diffusion types about the drug sustained release mechanism are suggested.

Effective Carrier-Concentration Tuning of SnO2 Quantum Dot Electron-Selective Layers for High-Performance Planar Perovskite Solar Cells.

The carrier concentration of the electron-selective layer (ESL) and hole-selective layer can significantly affect the performance of organic-inorganic lead halide perovskite solar cells (PSCs). Herein, a facile yet effective two-step method, i.e., room-temperature colloidal synthesis and low-temperature removal of additive (thiourea), to control the carrier concentration of SnO2 quantum dot (QD) ESLs to achieve high-performance PSCs is developed. By optimizing the electron density of SnO2 QD ESLs, a champion stabilized power output of 20.32% for the planar PSCs using triple cation perovskite absorber and 19.73% for those using CH3 NH3 PbI3 absorber is achieved. The superior uniformity of low-temperature processed SnO2 QD ESLs also enables the fabrication of ≈19% efficiency PSCs with an aperture area of 1.0 cm2 and 16.97% efficiency flexible device. The results demonstrate the promise of carrier-concentration-controlled SnO2 QD ESLs for fabricating stable, efficient, reproducible, large-scale, and flexible planar PSCs.

Myasthenia gravis and chronic inflammatory demyelinating polyneuropathy in the same patient - a case report.

PURPOSE: To investigate the clinical character, diagnosis and treatment of chronic inflammatory demyelinating polyneuropathy accompanying myasthenia gravis so as to improve the understanding of such diseases. MATERIALS AND METHODS: A case of chronic inflammatory demyelinating polyneuropathy combined with myasthenia gravis were analyzed retrospectively with review of the literature. RESULTS: This man was presented with chronic progressive sensory symptoms, flaccid tetraparesis, areflexia and protein-cell dissociation of cerebrospinal fluid. Nerve conduction study was indicative of demyelinating neuropathy. He was suspected as chronic inflammatory demyelinating polyneuropathy and treated with high-dose glucocorticoids. However, his condition worsened. Four months later, he was admitted and was diagnosed as combination of chronic inflammatory demyelinating polyneuropathy and myasthenia gravis. Good clinical results were observed after he was treated with pyridostigmine bromide, prednisone and mycophenolate mofetil. CONCLUSIONS: This case warns clinicians to be aware of these two diseases presenting in the same patient, and the possible implications on treatment choices. A common immunological abnormality might exist in this rare association, but it still remains unknown.

MgO Nanoparticle Modified Anode for Highly Efficient SnO2-Based Planar Perovskite Solar Cells.

Reducing the energy loss and retarding the carrier recombination at the interface are crucial to improve the performance of the perovskite solar cell (PSCs). However, little is known about the recombination mechanism at the interface of anode and SnO2 electron transfer layer (ETL). In this work, an ultrathin wide bandgap dielectric MgO nanolayer is incorporated between SnO2:F (FTO) electrode and SnO2 ETL of planar PSCs, realizing enhanced electron transporting and hole blocking properties. With the use of this electrode modifier, a power conversion efficiency of 18.23% is demonstrated, an 11% increment compared with that without MgO modifier. These improvements are attributed to the better properties of MgO-modified FTO/SnO2 as compared to FTO/SnO2, such as smoother surface, less FTO surface defects due to MgO passivation, and suppressed electron-hole recombinations. Also, MgO nanolayer with lower valance band minimum level played a better role in hole blocking. When FTO is replaced with Sn-doped In2O3 (ITO), a higher power conversion efficiency of 18.82% is demonstrated. As a result, the device with the MgO hole-blocking layer exhibits a remarkable improvement of all J-V parameters. This work presents a new direction to improve the performance of the PSCs based on SnO2 ETL by transparent conductive electrode surface modification.

A Pipeline for Studying and Engineering Single-Subunit Oligosaccharyltransferases.

Asparagine-linked (N-linked) protein glycosylation is one of the most abundant types of posttranslational modification, occurring in all domains of life. The central enzyme in N-linked glycosylation is the oligosaccharyltransferase (OST), which catalyzes the covalent attachment of preassembled glycans to specific asparagine residues in target proteins. Whereas in higher eukaryotes the OST is comprised of eight different membrane proteins, of which the catalytic subunit is STT3, in kinetoplastids and prokaryotes the OST is a monomeric enzyme bearing homology to STT3. Given their relative simplicity, these single-subunit OSTs (ssOSTs) have emerged as important targets for mechanistic dissection of poorly understood aspects of N-glycosylation and at the same time hold great potential for the biosynthesis of custom glycoproteins. To take advantage of this utility, this chapter describes a multipronged approach for studying and engineering ssOSTs that integrates in vivo screening technology with in vitro characterization methods, thereby creating a versatile and readily adaptable pipeline for virtually any ssOST of interest.

Low-Cost Carbazole-Based Hole-Transport Material for Highly Efficient Perovskite Solar Cells.

A low-cost carbazole-based small-molecule material, 1,3,6,8-tetra(N,N-p-dimethoxyphenylamino)-9-ethylcarbazole, was designed and synthesized through a facile three-step synthetic route. The material was characterized and applied as a hole-transport material (HTM) for low-temperature-processed planar perovskite solar cells (PSCs). Devices based on this new HTM exhibit a high power-conversion efficiency of 17.8 % that is comparable to that of PSCs based on the costly 2,2',7,7'-tetrakis[N,N-di(4-methoxyphenyl)amino]-9,9'-spirobifluorene (Spiro-OMeTAD) (18.6 %) .

Dopant-Free Hole-Transport Materials Based on Methoxytriphenylamine-Substituted Indacenodithienothiophene for Solution-Processed Perovskite Solar Cells.

Solution-processed hole transporting materials (HTMs) that are dopant-free show promise for use in low-cost, high-performance perovskite solar cells (PSCs). The highest-efficiency PSCs use organic HTMs, many of which have low mobilities and therefore require doping, which lowers the device stability. Additionally, these materials are not easily scaled because they often require complicated synthesis. Two new HTMs (IDT-TPA and IDTT-TPA) were synthesized, which contained either an extended fused-ring indacenodithiophene (IDT) or indacenodithienothiophene (IDTT) core and strong electron-donating methoxytriphenylamine (TPA) groups as the end-capping units. The extended conjugation in the backbone of IDTT-TPA resulted in stronger π-π interactions (3.321 Å) and a higher hole mobility of 6.46×10-4  cm2 V-1 s-1 when compared with that of IDT-TPA (9.53×10-5  cm2 V-1 s-1 ). A dopant-free, planar PSC that contained IDTT-TPA was fabricated and exhibited a high power conversion efficiency (PCE) of 15.7 %. This cell exhibited a higher PCE and less hysteresis than devices that contained IDT-TPA.

Reducing Hysteresis and Enhancing Performance of Perovskite Solar Cells Using Low-Temperature Processed Y-Doped SnO2 Nanosheets as Electron Selective Layers.

Despite the rapid increase of efficiency, perovskite solar cells (PSCs) still face some challenges, one of which is the current-voltage hysteresis. Herein, it is reported that yttrium-doped tin dioxide (Y-SnO2 ) electron selective layer (ESL) synthesized by an in situ hydrothermal growth process at 95 °C can significantly reduce the hysteresis and improve the performance of PSCs. Comparison studies reveal two main effects of Y doping of SnO2 ESLs: (1) it promotes the formation of well-aligned and more homogeneous distribution of SnO2 nanosheet arrays (NSAs), which allows better perovskite infiltration, better contacts of perovskite with SnO2 nanosheets, and improves electron transfer from perovskite to ESL; (2) it enlarges the band gap and upshifts the band energy levels, resulting in better energy level alignment with perovskite and reduced charge recombination at NSA/perovskite interfaces. As a result, PSCs using Y-SnO2 NSA ESLs exhibit much less hysteresis and better performance compared with the cells using pristine SnO2 NSA ESLs. The champion cell using Y-SnO2 NSA ESL achieves a photovoltaic conversion efficiency of 17.29% (16.97%) when measured under reverse (forward) voltage scanning and a steady-state efficiency of 16.25%. The results suggest that low-temperature hydrothermal-synthesized Y-SnO2 NSA is a promising ESL for fabricating efficient and hysteresis-less PSC.

Cytomembrane ATP-sensitive K+ channels in neurovascular unit targets of ischemic stroke in the recovery period.

The present study was to analyze the mechanism of cytomembrane ATP-sensitive K+ channels (KATP) in the neurovascular unit treatment of ischemic stroke in the recovery period. A total of 24 healthy adult male Wistar rats of 5-8 weeks age, weighing 160-200 g were randomly divided into the control (sham-operation group), model, KATP blocker and KATP opener groups (n=6 rats per group). Nylon cerebral artery occlusion was conducted using nylon monofilament coated with Poly-L-lysine, which was used to produce a cerebral infarction model. After feeding normally for 3 days, 5-hydroxydecanoate (40 mg/Kg), and diazoxide (40 mg/Kg) were injected to the abdominal cavity in the blocker, and opener groups, respectively. The control received an equivalent normal saline that was injected into the sham-operation and model groups. The animals were mutilated and samples were collected after 3 days. RT-PCR was used to detect the expression levels of the three subunits of KATP, i.e., kir6.1, and sulfonylurea receptor (SUR) 1 and SUR2 mRNA, as well as to calculate infarct size in tetrazolium chloride staining. The expression level of mRNA in the opener group were significantly higher, followed by the model and blocker groups, with the control group being the lowest (P<0.05). Infarct size in the opener group was markedly smaller than the model and blocker groups, and infarct size in the blocker group was significantly larger (P<0.05). Thus, the target treatment on KATP may improve the prognosis of ischemic stroke during the recovery period.