Synthesis of tertiary alkylphosphonate oligonucleotides through light-driven radical-polar crossover reactions.

Chemical modification of nucleotides can improve the metabolic stability and target specificity of oligonucleotide therapeutics, and alkylphosphonates have been employed as charge-neutral replacements for naturally-occurring phosphodiester backbones in these compounds. However, at present, the alkyl moieties that can be attached to phosphorus atoms in these compounds are limited to methyl groups or primary/secondary alkyls, and such alkylphosphonate moieties can degrade during oligonucleotide synthesis. The present work demonstrates the tertiary alkylation of the phosphorus atoms of phosphites bearing two 2'-deoxynuclosides. This process utilizes a carbocation generated via a light-driven radical-polar crossover mechanism. This protocol provides tertiary alkylphosphonate structures that are difficult to synthesize using existing methods. The conversion of these species to oligonucleotides having charge-neutral alkylphosphonate linkages through a phosphoramidite-based approach was also confirmed in this study.

LC-MS/MS quantitation of elastin crosslinker desmosines and histological analysis of skin aging characteristics in mice.

Elastic fibers consist of an insoluble inner core of elastin, which confers elasticity and resilience to vertebral organs and tissues. Desmosine (DES) and isodesmosine (IDES) are potential biomarkers of pathologies that lead to decreased elastin turnover. Mice are commonly used in research to mimic humans because of their similar genetics, physiology, and organ systems. The present study thus used senescent accelerated prone (SAMP10) and senescent accelerated resistant (SAMR1) mice to examine the connection between aging and histological or biomolecular changes. Mice were divided into three groups: SAMP10 fed a control diet (CD), SAMP10 fed a high-fat diet (HFD), and SAMR1 fed a CD. The percent liver to total body weight ratio (%LW/BW), desmosines (DESs or DES/IDES) content, and histological alterations in skin samples were evaluated. DESs were quantified using an isotope-dilution liquid chromatography-tandem mass spectrometry method with isodesmosine-13C3,15N1 as the internal standard (ISTD). The assays were repeatable, reproducible, and accurate, with %CV values ≤ (1.90, 1.77, and 3.03), ISTD area %RSD of (1.54, 0.92, and 1.13), and %AC of (99.02 ± 1.86, 101.00 ± 2.30, and 101.30 ± 2.90) for the calibrations (equimolar DES/IDES, DES, and IDES, respectively). The average DESs content per dry-weight abdominal skin and %LW/BW were similar between the three groups. Histological analyses revealed elastin fibers in five randomly selected samples. The epidermis and dermal white adipose tissue layers were thicker in SAMP10 mice than SAMR1 mice. Thus, characteristic signs of aging in SAMP10 and SAMR1 mice could not be differentiated based on measurement of DESs content of the skin or %LW/BW, but aging could be differentiated based on microscopic analysis of histological changes in the skin components of SAMP10 and SAMR1 mice.

Discovery of a Novel Series of Potent, Selective, Orally Available, and Brain-Penetrable C1s Inhibitors for Modulation of the Complement Pathway.

A novel series of non-amidine-based C1s inhibitors have been explored. Starting from high-throughput screening hit 3, isoquinoline was replaced with 1-aminophthalazine to enhance C1s inhibitory activity while exhibiting good selectivity against other serine proteases. We first disclose a crystal structure of a complex of C1s and a small-molecule inhibitor (4e), which guided structure-based optimization around the S2 and S3 sites to further enhance C1s inhibitory activity by over 300-fold. Improvement of membrane permeability by incorporation of fluorine at the 8-position of 1-aminophthalazine led to identification of (R)-8 as a potent, selective, orally available, and brain-penetrable C1s inhibitor. (R)-8 significantly inhibited membrane attack complex formation induced by human serum in a dose-dependent manner in an in vitro assay system, proving that selective C1s inhibition blocked the classical complement pathway effectively. As a result, (R)-8 emerged as a valuable tool compound for both in vitro and in vivo assessment.

Photoredox-based late-stage functionalization in SAR study for in vivo potent glucosylceramide synthase inhibitor.

Glucosylceramide synthase (GCS) has drawn much attention as an attractive protein target in the disease pathways of Parkinson's Disease (PD) and lysosomal storage disorders, such as Gaucher's Disease (GD). In previous our study, T-036 and its analogue, 2a, were discovered as novel GCS inhibitors. To further improve activity of this chemical series, SAR was investigated on the fused pyridyl ring core of 2a by employing a photoredox reaction that significantly reduced synthetic demand. Herein, we successfully applied the decarboxylation C-H alkylation photoredox reaction to introduce a wide variety of substituents at the 6-position of the fused pyridine core scaffold. This quick SAR acquisition facilitated the swift identification of the potent GCS inhibitors 2b (IC50 = 5.9 nM) and 2g (IC50 = 3.6 nM). Moreover, 2b exhibited superior in vivo potency to that of our previously reported lead compound, T-036.

Study protocol for HGCSG1801: A multicenter, prospective, phase II trial of second-line FOLFIRI plus aflibercept in patients with metastatic colorectal cancer refractory to anti-EGFR antibodies.

Background: The first-line chemotherapy for patients with RAS and BRAF wild-type metastatic colorectal cancer (mCRC) commonly involves cytotoxic regimens, such as FOLFOX and FOLFIRI, combined with epidermal growth factor receptor (EGFR) antibodies. When progression occurs following anti-EGFR antibody-combined chemotherapy, anti-angiogenic inhibitors can be used as second-line treatment. Although randomized controlled trials have shown that anti-angiogenic inhibitors [bevacizumab, ramucirumab, and aflibercept (AFL)] carry survival benefit when combined with FOLFIRI as second-line chemotherapy, such trials did not provide data on patients with mCRC refractory to anti-EGFR antibody-combined chemotherapy. Therefore, our group planned a multicenter, nonrandomized, single-arm, prospective, phase II study to investigate the safety and efficacy of FOLFIRI plus AFL as a second-line chemotherapy for patients with mCRC refractory to oxaliplatin-based chemotherapy combined with anti-EGFR antibodies. Methods: FOLFIRI (irinotecan 180 mg/m2, l-leucovorin 200 mg/m2, bolus 5-FU 400 mg/m2, and infusional 5-FU 2400 mg/m2/46 h) and AFL (4 mg/kg) will be administered every 2 weeks until progression or unacceptable toxicities occur. The primary endpoint will be the 6-month progression-free survival (PFS) rate, whereas the secondary endpoints will include overall survival, PFS, response rate, disease control rate, adverse events, and relative dose intensity for each drug. A sample size of 41 participants will be required. This study will be sponsored by the Non-Profit Organization Hokkaido Gastrointestinal Cancer Study Group and will be supported by a grant from Sanofi. Discussion: There is only an observational study reporting data on FOLFIRI plus AFL for patients with mCRC who previously received anti-EGFR antibodies; therefore, a prospective clinical trial is needed. This study will prospectively evaluate the efficacy and safety of FOLFIRI plus AFL in patients with mCRC who are resistant to anti-EGFR antibodies and have limited data. Moreover, this study will reveal predictive biomarkers for AFL-based chemotherapy. Clinical trial registration: Japan Registry of Clinical Trials, jRCTs011190006. Registered 19 November, 2019, https://jrct.niph.go.jp/latest-detail/jRCTs011190006.

A Chemical Strategy toward Novel Brain-Penetrant EZH2 Inhibitors.

Aberrant gene-silencing through dysregulation of polycomb protein activity has emerged as an important oncogenic mechanism in cancer, implicating polycomb proteins as important therapeutic targets. Recently, an inhibitor targeting EZH2, the methyltransferase component of PRC2, received U.S. Food and Drug Administration approval following promising clinical responses in cancer patients. However, the current array of EZH2 inhibitors have poor brain penetrance, limiting their use in patients with central nervous system malignancies, a number of which have been shown to be sensitive to EZH2 inhibition. To address this need, we have identified a chemical strategy, based on computational modeling of pyridone-containing EZH2 inhibitor scaffolds, to minimize P-glycoprotein activity, and here we report the first brain-penetrant EZH2 inhibitor, TDI-6118 (compound 5). Additionally, in the course of our attempts to optimize this compound, we discovered TDI-11904 (compound 21), a novel, highly potent, and peripherally active EZH2 inhibitor based on a 7 member ring structure.

Selective PPARα Modulator Pemafibrate and Sodium-Glucose Cotransporter 2 Inhibitor Tofogliflozin Combination Treatment Improved Histopathology in Experimental Mice Model of Non-Alcoholic Steatohepatitis.

Ballooning degeneration of hepatocytes is a major distinguishing histological feature of non-alcoholic steatosis (NASH) progression that can lead to cirrhosis and hepatocellular carcinoma (HCC). In this study, we evaluated the effect of the selective PPARα modulator (SPPARMα) pemafibrate (Pema) and sodium-glucose cotransporter 2 (SGLT2) inhibitor tofogliflozin (Tofo) combination treatment on pathological progression in the liver of a mouse model of NASH (STAM) at two time points (onset of NASH progression and HCC survival). At both time points, the Pema and Tofo combination treatment significantly alleviated hyperglycemia and hypertriglyceridemia. The combination treatment significantly reduced ballooning degeneration of hepatocytes. RNA-seq analysis suggested that Pema and Tofo combination treatment resulted in an increase in glyceroneogenesis, triglyceride (TG) uptake, lipolysis and liberated fatty acids re-esterification into TG, lipid droplet (LD) formation, and Cidea/Cidec ratio along with an increased number and reduced size and area of LDs. In addition, combination treatment reduced expression levels of endoplasmic reticulum stress-related genes (Ire1a, Grp78, Xbp1, and Phlda3). Pema and Tofo treatment significantly improved survival rates and reduced the number of tumors in the liver compared to the NASH control group. These results suggest that SPPARMα and SGLT2 inhibitor combination therapy has therapeutic potential to prevent NASH-HCC progression.

Metabolic engineering for valorization of macroalgae biomass.

Marine macroalgae have huge potential as feedstocks for production of a wide spectrum of chemicals used in biofuels, biomaterials, and bioactive compounds. Harnessing macroalgae in these ways could promote wellbeing for people while mitigating climate change and environmental destruction linked to use of fossil fuels. Microorganisms play pivotal roles in converting macroalgae into valuable products, and metabolic engineering technologies have been developed to extend their native capabilities. This review showcases current achievements in engineering the metabolisms of various microbial chassis to convert red, green, and brown macroalgae into bioproducts. Unique features of macroalgae, such as seasonal variation in carbohydrate content and salinity, provide the next challenges to advancing macroalgae-based biorefineries. Three emerging engineering strategies are discussed here: (1) designing dynamic control of metabolic pathways, (2) engineering strains of halophilic (salt-tolerant) microbes, and (3) developing microbial consortia for conversion. This review illuminates opportunities for future research communities by elucidating current approaches to engineering microbes so they can become cell factories for the utilization of macroalgae feedstocks.

Assessment of an Automatic Hot Water Sterilizing System-Equipped Dialysis Supply Device's Circuit Degradation, 7 Years Postinstallment.

At most dialysis clinics in Japan, a central dialysis fluid delivery system (CDDS) is used primarily to provide consistent treatment to patients. We incorporated hot water disinfection to a CDDS over a 7 year period, after which we assessed the dialysis purification levels. We observed that adequate levels had been maintained. We rated the internal circuits of the device at the 7 year mark, comparing the insides of both the used and unused new Synflex tubing, silicone blades, Teflon tubing, silicone rubber tubing, and stainless steel parts. No bacteria or contamination (such as endotoxins) was detected, and no degradation or damage were observed. Even after the auto-hot water disinfection system was installed, no internal tubing degradation due to hot water or mechanical issues have occurred, and the system's dialysis fluid purification levels have been maintained.

Genomics Characterization of an Engineered Corynebacterium glutamicum in Bioreactor Cultivation Under Ionic Liquid Stress.

Corynebacterium glutamicum is an ideal microbial chassis for production of valuable bioproducts including amino acids and next generation biofuels. Here we resequence engineered isopentenol (IP) producing C. glutamicum BRC-JBEI 1.1.2 strain and assess differential transcriptional profiles using RNA sequencing under industrially relevant conditions including scale transition and compare the presence vs absence of an ionic liquid, cholinium lysinate ([Ch][Lys]). Analysis of the scale transition from shake flask to bioreactor with transcriptomics identified a distinct pattern of metabolic and regulatory responses needed for growth in this industrial format. These differential changes in gene expression corroborate altered accumulation of organic acids and bioproducts, including succinate, acetate, and acetoin that occur when cells are grown in the presence of 50 mM [Ch][Lys] in the stirred-tank reactor. This new genome assembly and differential expression analysis of cells grown in a stirred tank bioreactor clarify the cell response of an C. glutamicum strain engineered to produce IP.

Decarboxylative N-Alkylation of Azoles through Visible-Light-Mediated Organophotoredox Catalysis.

An organophotoredox-catalyzed decarboxylative cross-coupling between azole nucleophiles and aliphatic carboxylic acid-derived redox-active esters is demonstrated. This protocol efficiently installs various tertiary or secondary alkyl fragments onto the nitrogen atom of azole nucleophiles under mild and transition-metal-free conditions. The pyridinium additive successfully inhibits the formation of elimination byproducts from the carbocation intermediate. This reaction is applicable to the synthesis of a protein-degrader-like molecule containing an azole and a thalidomide.

Effect of Percentage of Femoral Anterior Cruciate Ligament Insertion Site Reconstructed With Hamstring Tendon on Knee Kinematics and Graft Force.

BACKGROUND: Previous studies have stated that closely matching the size of the anterior cruciate ligament (ACL) insertion site footprint is important for biomechanical function and clinical stability after ACL reconstruction. However, the ACL varies widely regarding the area of femoral insertion, tibial insertion, and midsubstance of ACL, and reconstructing the insertion site area with a uniform diameter graft can result in a cross-sectional area that is greater than that of the midsubstance of the native ACL. Therefore, understanding the effect of relative graft size in ACL reconstruction on knee biomechanics is important for surgical planning. PURPOSE: To assess how the percentage of femoral insertion site affects knee biomechanics in single- and double-bundle ACL reconstruction. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 14 human cadaveric knees were scanned with magnetic resonance imaging and tested using a robotic system under an anterior tibial load and a combined rotational load. In total, 7 knee states were evaluated: intact ACL; deficient ACL; single-bundle ACL reconstruction with approximate graft sizes 25% (small), 50% (medium), and 75% (large) of the femoral insertion site; and double-bundle reconstruction of approximately 50% (medium) and 75% (large) of the femoral insertion site, based on the ratio of the cross-sectional area of the graft to the area of the femoral ACL insertion site determined by magnetic resonance imaging. RESULTS: Anterior tibial translation was not significantly larger than the intact state in single-bundle and double-bundle medium graft reconstructions (P > .05) and was significantly greater in the single-bundle small graft reconstruction (P < .05). Anterior knee translation in single-bundle medium graft and large graft reconstructions was not statistically different (P > .05). In contrast, the anterior tibial translation for double-bundle large graft reconstruction was significantly smaller than for double-bundle medium graft reconstruction at low flexion angles (P < .05). The single-bundle small graft force was significantly different from the intact ACL in situ force (P < .05). The graft force with double-bundle large reconstruction was significantly greater than that with the double-bundle medium reconstruction (P < .05) but was not significantly different from that of the intact ACL (P > .05). CONCLUSION: Knee biomechanics with a single-bundle small graft tended to be significantly different from those of the intact knee. In the kinematic and kinetic data for the single- and double-bundle medium graft reconstruction, only the anterior translation at full extension for the single-bundle reconstruction was significantly different (lower) from that of intact knee. This was a time zero study. CLINICAL RELEVANCE: This study can provide surgeons with guidance in selecting the graft size for ACL reconstruction.

Multisession CyberKnife Radiosurgery for Orbital Cavernous Hemangiomas.

Orbital cavernous hemangioma is a rare benign tumor that causes protrusion of the eye-ball and lowers visual acuity. Surgical resection is the first choice of treatment, however, it is challenging due to the anatomical structure of the orbit. Stereotactic radiosurgery (SRS) has become an established alternative treatment for any challenging microsurgery in the skull. Five cases of orbital cavernous hemangioma were successfully treated with multisession CyberKnife (Accuray, Sunnyvale, CA, USA) stereotactic radiotherapy. The tumor volume ranged from 0.2 cc to 11.9 cc, all of which decreased in size after treatment. The radiation prescription dose ranged from 24 Gy to 26.1 Gy with fractions of 5 or 6. Visual acuity was preserved in all cases. Thus, multisession CyberKnife radiotherapy for orbital cavernous hemangioma might be a safe and effective treatment option.

Hemostasis through Extended Transsphenoidal Route for Subarachnoid Hemorrhage after Conventional Transsphenoidal Surgery for a Pituitary Adenoma.

This report describes a 49-year-old male patient who presented with a pituitary adenoma extending to the suprasellar region. Subarachnoid hemorrhage (SAH) occurred after conventional transnasal transsphenoidal surgery for a non-functioning pituitary adenoma despite no suprasellar arachnoid membrane breakdown. Through extended transsphenoidal route, the suprasellar hematoma was removed and bleeding from a small vessel thought to be the branch of left superior hypophyseal artery was successfully controlled. Indeed, several case reports regarding this rare complication have been published, but the mechanism of SAH has never been identified and the prognosis was poor in most cases. This report illustrates the origin and mechanism of the bleeding clearly using the intraoperative video. This case suggests that immediate therapeutic intervention is necessary and extended transnasal transsphenoidal repeat surgery is useful for an appropriate hemostasis and removal of hematoma located in the suprasellar region.

Differential mode delay diagnostics for LP modes traversing a few-mode fiber.

We provide differential mode delay (DMD) diagnostics for linearly polarized (LP) modes traversing a few-mode fiber (FMF). We employ a modal interferometer method with a butt coupling mechanism to measure the DMD between any two modes guided in FMFs with step-index (SI) and depressed-cladding profiles. The measurement principle is based on investigating a transmitted spectrum through temporal decomposition by means of a Fourier transform. At least four different temporal waveforms are needed for an FMF supporting four LP modes to estimate the DMD between two different LP modes. A butt coupling mechanism with an air gap between an FMF and a dispersion-shifted fiber is employed for simultaneous DMD measurement. The present diagnostics requires some knowledge of the bending loss characteristics of the FMFs and the butt coupling properties of the circular symmetry modes or circular asymmetry modes in their electric fields. The DMD diagnostics are realized taking the bending loss and butt coupling characteristics into consideration. We present experimental results for the DMD and DMD slope as a function of the wavelength in a 1450-1625 nm telecommunication band.

CyberKnife Radiotherapy for Skull Base Petroclival Metastases Including Dorello's Canal: Report of 10 Cases.

Skull base petroclival metastases cause diplopia due to abducens nerve palsy. Diplopia is visually disabling, and skull base metastasis is extremely difficult to treat even with microscopic surgery. However, stereotactic radiotherapy with CyberKnife (Accuray Incorporated, Sunnyvale, California) has been very successful in 10 cases. As the abducens nerve runs through Dorello's canal in the skull base, the radiation dose and fraction were adjusted to avoid damage to the nerve. Since these metastases are not located inside the brain but in the skull base, contrast magnetic resonance imaging (MRI) combined with fluorodeoxyglucose-positron emission tomography (FDG-PET) was essential to detect the cancers.

Genome-scale metabolic rewiring improves titers rates and yields of the non-native product indigoidine at scale.

High titer, rate, yield (TRY), and scalability are challenging metrics to achieve due to trade-offs between carbon use for growth and production. To achieve these metrics, we take the minimal cut set (MCS) approach that predicts metabolic reactions for elimination to couple metabolite production strongly with growth. We compute MCS solution-sets for a non-native product indigoidine, a sustainable pigment, in Pseudomonas putida KT2440, an emerging industrial microbe. From the 63 solution-sets, our omics guided process identifies one experimentally feasible solution requiring 14 simultaneous reaction interventions. We implement a total of 14 genes knockdowns using multiplex-CRISPRi. MCS-based solution shifts production from stationary to exponential phase. We achieve 25.6 g/L, 0.22 g/l/h, and ~50% maximum theoretical yield (0.33 g indigoidine/g glucose). These phenotypes are maintained from batch to fed-batch mode, and across scales (100-ml shake flasks, 250-ml ambr®, and 2-L bioreactors).

Long-range intramolecular allostery and regulation in the dynein-like AAA protein Mdn1.

Mdn1 is an essential mechanoenzyme that uses the energy from ATP hydrolysis to physically reshape and remodel, and thus mature, the 60S subunit of the ribosome. This massive (>500 kDa) protein has an N-terminal AAA (ATPase associated with diverse cellular activities) ring, which, like dynein, has six ATPase sites. The AAA ring is followed by large (>2,000 aa) linking domains that include an ∼500-aa disordered (D/E-rich) region, and a C-terminal substrate-binding MIDAS domain. Recent models suggest that intramolecular docking of the MIDAS domain onto the AAA ring is required for Mdn1 to transmit force to its ribosomal substrates, but it is not currently understood what role the linking domains play, or why tethering the MIDAS domain to the AAA ring is required for protein function. Here, we use chemical probes, single-particle electron microscopy, and native mass spectrometry to study the AAA and MIDAS domains separately or in combination. We find that Mdn1 lacking the D/E-rich and MIDAS domains retains ATP and chemical probe binding activities. Free MIDAS domain can bind to the AAA ring of this construct in a stereo-specific bimolecular interaction, and, interestingly, this binding reduces ATPase activity. Whereas intramolecular MIDAS docking appears to require a treatment with a chemical inhibitor or preribosome binding, bimolecular MIDAS docking does not. Hence, tethering the MIDAS domain to the AAA ring serves to prevent, rather than promote, MIDAS docking in the absence of inducing signals.

Pemafibrate, a selective PPARα modulator, prevents non-alcoholic steatohepatitis development without reducing the hepatic triglyceride content.

Non-alcoholic steatohepatitis (NASH) is characterized by macrovesicular steatosis with ballooning degeneration of hepatocytes, diffused lobular inflammation, and fibrosis. PPAR ligands are promising therapeutic agents in NASH; accordingly, we evaluated the effects of the first clinically available selective PPARα modulator, pemafibrate. We found that pemafibrate improves F4/80-positive macrophage accumulation, ballooning degeneration of hepatocytes, and the non-alcoholic fatty liver disease (NAFLD) activity score without affecting triglyceride (TG) accumulation in the liver of a mouse model of NASH (STAM). A global gene expression analysis indicated that pemafibrate enhances TG hydrolysis and fatty acid ß-oxidation as well as re-esterification from dihydroxyacetone 3-phosphate and monoacylglycerol to TG. These changes are accompanied by the induction of genes involved in lipolysis and lipid droplet formation, along with an increased number and reduced size of lipid droplets in pemafibrate-treated livers. Pemafibrate reduced the expression of the cell adhesion molecule Vcam-1, myeloid cell markers, and inflammation- and fibrosis-related genes in STAM mice. Furthermore, pemafibrate significantly reduced VCAM-1 expression induced by high glucose in cultured human umbilical vein endothelial cells. These results suggest that pemafibrate prevents NASH development by reducing myeloid cell recruitment via interactions with liver sinusoidal endothelial cells, without altering hepatic TG accumulation.

Efficient synthesis of tert-butyl 3-cyano-3-cyclopropyl-2-oxopyrrolidine-4-carboxylates: Highly functionalized 2-pyrrolidinone enabling access to novel macrocyclic Tyk2 inhibitors.

Herein we report an efficient method for the synthesis of a highly functionalized 2-pyrrolidinone, tert-butyl 3-cyano-3-cyclopropyl-2-oxopyrrolidine-4-carboxylate, from readily available starting materials. Utility of this compound was demonstrated in the synthesis of a novel series of macrocyclic Tyk2 inhibitors, leading to the identification of a potent and selective macrocyclic Tyk2 inhibitor (26).