Targeting a lineage-specific PI3KÉ£-Akt signaling module in acute myeloid leukemia using a heterobifunctional degrader molecule.

Dose-limiting toxicity poses a major limitation to the clinical utility of targeted cancer therapies, often arising from target engagement in nonmalignant tissues. This obstacle can be minimized by targeting cancer dependencies driven by proteins with tissue-restricted and/or tumor-restricted expression. In line with another recent report, we show here that, in acute myeloid leukemia (AML), suppression of the myeloid-restricted PIK3CG/p110γ-PIK3R5/p101 axis inhibits protein kinase B/Akt signaling and compromises AML cell fitness. Furthermore, silencing the genes encoding PIK3CG/p110γ or PIK3R5/p101 sensitizes AML cells to established AML therapies. Importantly, we find that existing small-molecule inhibitors against PIK3CG are insufficient to achieve a sustained long-term antileukemic effect. To address this concern, we developed a proteolysis-targeting chimera (PROTAC) heterobifunctional molecule that specifically degrades PIK3CG and potently suppresses AML progression alone and in combination with venetoclax in human AML cell lines, primary samples from patients with AML and syngeneic mouse models.

Diversity of mitochondrial DNA in 3 species of great whales before and after modern whaling.

The 20th century commercial whaling industry severely reduced populations of great whales throughout the Southern Hemisphere. The effect of this exploitation on genetic diversity and population structure remains largely undescribed. Here, we compare pre- and post-whaling diversity of mitochondrial DNA (mtDNA) control region sequences for 3 great whales in the South Atlantic, such as the blue, humpback, and fin whale. Pre-whaling diversity is described from mtDNA extracted from bones collected near abandoned whaling stations, primarily from the South Atlantic island of South Georgia. These bones are known to represent the first stage of 20th century whaling and thus pre-whaling diversity of these populations. Post-whaling diversity is described from previously published studies reporting large-scale sampling of living whales in the Southern Hemisphere. Despite relatively high levels of surviving genetic diversity in the post-whaling populations, we found evidence of a probable loss of mtDNA lineages in all 3 species. This is evidenced by the detection of a large number of haplotypes found in the pre-whaling samples that are not present in the post-whaling samples. A rarefaction analysis further supports a loss of haplotypes in the South Atlantic humpback and Antarctic blue whale populations. The bones from former whaling stations in the South Atlantic represent a remarkable molecular archive for further investigation of the decline and ongoing recovery in the great whales of the Southern Hemisphere.

Covalent-reversible peptide-based protease inhibitors. Design, synthesis, and clinical success stories.

Dysregulated human peptidases are implicated in a large variety of diseases such as cancer, hypertension, and neurodegeneration. Viral proteases for their part are crucial for the pathogens' maturation and assembly. Several decades of research were devoted to exploring these precious therapeutic targets, often addressing them with synthetic substrate-based inhibitors to elucidate their biological roles and develop medications. The rational design of peptide-based inhibitors offered a rapid pathway to obtain a variety of research tools and drug candidates. Non-covalent modifiers were historically the first choice for protease inhibition due to their reversible enzyme binding mode and thus presumably safer profile. However, in recent years, covalent-irreversible inhibitors are having a resurgence with dramatic increase of their related publications, preclinical and clinical trials, and FDA-approved drugs. Depending on the context, covalent modifiers could provide more effective and selective drug candidates, hence requiring lower doses, thereby limiting off-target effects. Additionally, such molecules seem more suitable to tackle the crucial issue of cancer and viral drug resistances. At the frontier of reversible and irreversible based inhibitors, a new drug class, the covalent-reversible peptide-based inhibitors, has emerged with the FDA approval of Bortezomib in 2003, shortly followed by 4 other listings to date. The highlight in the field is the breathtakingly fast development of the first oral COVID-19 medication, Nirmatrelvir. Covalent-reversible inhibitors can hipothetically provide the safety of the reversible modifiers combined with the high potency and specificity of their irreversible counterparts. Herein, we will present the main groups of covalent-reversible peptide-based inhibitors, focusing on their design, synthesis, and successful drug development programs.

MCB-613 exploits a collateral sensitivity in drug resistant EGFR-mutant non-small cell lung cancer through covalent inhibition of KEAP1.

Targeted therapies have revolutionized cancer chemotherapy. Unfortunately, most patients develop multifocal resistance to these drugs within a matter of months. Here, we used a high-throughput phenotypic small molecule screen to identify MCB-613 as a compound that selectively targets EGFR-mutant, EGFR inhibitor-resistant non-small cell lung cancer (NSCLC) cells harboring diverse resistance mechanisms. Subsequent proteomic and functional genomic screens involving MCB-613 identified its target in this context to be KEAP1, revealing that this gene is selectively essential in the setting of EGFR inhibitor resistance. In-depth molecular characterization demonstrated that (1) MCB-613 binds KEAP1 covalently; (2) a single molecule of MCB-613 is capable of bridging two KEAP1 monomers together; and, (3) this modification interferes with the degradation of canonical KEAP1 substrates such as NRF2. Surprisingly, NRF2 knockout sensitizes cells to MCB-613, suggesting that the drug functions through modulation of an alternative KEAP1 substrate. Together, these findings advance MCB-613 as a new tool for exploiting the selective essentiality of KEAP1 in drug-resistant, EGFR-mutant NSCLC cells.

Radiographic Outcomes following K-Wire Augmentation of Dorsal Spanning Plate Fixation for Intra-Articular Fractures of the Distal Radius.

Background Restoration of articular surface alignment is critical in treating intra-articular distal radius fractures. Dorsal spanning plate fixation functions as an internal distraction mechanism and can be advantageous in the setting of highly comminuted fracture patterns, polytrauma patients, and patients with radiocarpal instability. The addition of K-wires to support articular surface reduction potentially augments fracture repair stability. Questions/Purposes We examined the radiographic outcomes and maintenance of reduction in patients with comminuted intra-articular distal radius fractures treated with K-wire fixation of articular fragments followed by dorsal spanning plate application. Patients and Methods We reviewed 35 consecutive patients with complex intra-articular distal radius fractures treated with dorsal spanning plate and K-wire fixation between April 2016 and October 2019. AO classification was recorded: B1 (3), B3 (2), C2 (2), C3 (28). A two-tailed paired t -test was used to compare findings immediately post-dorsal spanning plate surgery and at final follow-up after dorsal spanning plate removal. Results Mean patient age was 43.3 years (19-78 years). Mean follow-up was 7.8 months (SD 4.3 months) from surgery and 2.5 months from pin removal (SD 2.6 months). All patients achieved radiographic union. Radial height (mean interval change (MIC) 0.2 mm, SD 2.2, p = 0.63), articular step-off (MIC 0.1 mm, SD 0.6 mm, p = 0.88), and radial inclination (MIC -1.1 degrees, SD 3.7 degrees, p = 0.10) did not change from post-surgery to final follow-up. Ulnar variance (MIC -0.9 mm, SD 2.0 mm, p = 0.02) and volar tilt (MIC -1.5 degrees, SD 4.4 degrees, p = 0.05) were found to have decreased. Conclusion Dorsal spanning plate augmented with K-wire fixation for comminuted intra-articular distal radius fractures in polytrauma patients allows for immediate weightbearing and maintains articular surface alignment at radiographic union and may provide better articular restoration than treatment with dorsal spanning plate alone. Level of Evidence This is a Level IV , therapeutic study.

The Unexpected Helical Supramolecular Assembly of a Simple Achiral Acetamide Tecton Generates Selective Water Channels.

Invited for the cover of this issue is the collaborative research team coordinated by Arie van der Lee at the University of Montpellier. The image depicts chiral channels with highly mobile water molecules resulting from the robust self-organization of a simple achiral acetamide. Fully reversible release and re-uptake of water molecules takes place near ambient conditions, with efficient water transport and a good selectivity against NaCl suggesting it to be an efficient candidate for desalination processes. Read the full text of the article at 10.1002/chem.20200383.

The Unexpected Helical Supramolecular Assembly of a Simple Achiral Acetamide Tecton Generates Selective Water Channels.

Achiral 2-hydroxy-N-(diphenylmethyl)acetamide (HNDPA) crystallizes in the P61 chiral space group as a hydrate, building up permeable chiral crystalline helical water channels. The crystallization-driven chiral self-resolution process is highly robust, with the same air-stable crystalline form readily obtained under a variety of conditions. Interestingly, the HNDPA supramolecular helix inner pore is filled by a helical water wire. The whole edifice is mainly stabilized by robust hydrogen bonds involving the HNDPA amide bonds and CH… π interactions between the HNDPA phenyl groups. The crystalline structure shows breathing behavior, with completely reversible release and re-uptake of water inside the chiral channel under ambient conditions. Importantly, the HNDPA channel is able to transport water very efficiently and selectively under biomimetic conditions. With a permeability per channel of 3.3 million water molecules per second in large unilamellar vesicles (LUV) and total selectivity against NaCl, the HNDPA channel is a very promising functional nanomaterial for future applications.

Ultrasound-assisted one-pot three-component synthesis of new isoxazolines bearing sulfonamides and their evaluation against hematological malignancies.

In the present study, following a one-pot two-step protocol, we have synthesized novel sulfonamides-isoxazolines hybrids (3a-r) via a highly regioselective 1,3-dipolar cycloaddition. The present methodology capitalized on trichloroisocyanuric acid (TCCA) as a safe and ecological oxidant and chlorinating agent for the in-situ conversion of aldehydes to nitrile oxides in the presence of hydroxylamine hydrochloride, under ultrasound activation. These nitrile oxides could be engaged in 1,3-dipolar cycloaddition reactions with various alkene to afford the targeted sulfonamides-isoxazolines hybrids (3a-r). The latter were assessed for their antineoplastic activity against model leukemia cell lines (Chronic Myeloid Leukemia, K562 and Promyelocytic Leukemia, HL-60).

Dual Covalent Inhibition of PKM and IMPDH Targets Metabolism in Cutaneous Metastatic Melanoma.

Overcoming acquired drug resistance is a primary challenge in cancer treatment. Notably, more than 50% of patients with BRAFV600E cutaneous metastatic melanoma (CMM) eventually develop resistance to BRAF inhibitors. Resistant cells undergo metabolic reprogramming that profoundly influences therapeutic response and promotes tumor progression. Uncovering metabolic vulnerabilities could help suppress CMM tumor growth and overcome drug resistance. Here we identified a drug, HA344, that concomitantly targets two distinct metabolic hubs in cancer cells. HA344 inhibited the final and rate-limiting step of glycolysis through its covalent binding to the pyruvate kinase M2 (PKM2) enzyme, and it concurrently blocked the activity of inosine monophosphate dehydrogenase, the rate-limiting enzyme of de novo guanylate synthesis. As a consequence, HA344 efficiently targeted vemurafenib-sensitive and vemurafenib-resistant CMM cells and impaired CMM xenograft tumor growth in mice. In addition, HA344 acted synergistically with BRAF inhibitors on CMM cell lines in vitro. Thus, the mechanism of action of HA344 provides potential therapeutic avenues for patients with CMM and a broad range of different cancers. SIGNIFICANCE: Glycolytic and purine synthesis pathways are often deregulated in therapy-resistant tumors and can be targeted by the covalent inhibitor described in this study, suggesting its broad application for overcoming resistance in cancer.

Nanofibrous hyaluronic acid scaffolds delivering TGF-ß3 and SDF-1α for articular cartilage repair in a large animal model.

Focal cartilage injuries have poor intrinsic healing potential and often progress to osteoarthritis, a costly disease affecting almost a third of adults in the United States. To treat these patients, cartilage repair therapies often use cell-seeded scaffolds, which are limited by donor site morbidity, high costs, and poor efficacy. To address these limitations, we developed an electrospun cell-free fibrous hyaluronic acid (HA) scaffold that delivers factors specifically designed to enhance cartilage repair: Stromal Cell-Derived Factor-1α (SDF-1α; SDF) to increase the recruitment and infiltration of mesenchymal stem cells (MSCs) and Transforming Growth Factor-ß3 (TGF-ß3; TGF) to enhance cartilage tissue formation. Scaffolds were characterized in vitro and then deployed in a large animal model of full-thickness cartilage defect repair. The bioactivity of both factors was verified in vitro, with both SDF and TGF increasing cell migration, and TGF increasing matrix formation by MSCs. In vivo, however, scaffolds releasing SDF resulted in an inferior cartilage healing response (lower mechanics, lower ICRS II histology score) compared to scaffolds releasing TGF alone. These results highlight the importance of translation into large animal models to appropriately screen scaffolds and therapies, and will guide investigators towards alternative growth factor combinations. STATEMENT OF SIGNIFICANCE: This study addresses an area of orthopaedic medicine in which treatment options are limited and new biomaterials stand to improve patient outcomes. Those suffering from articular cartilage injuries are often destined to have early onset osteoarthritis. We have created a cell-free nanofibrous hyaluronic acid (HA) scaffold that delivers factors specifically designed to enhance cartilage repair: Stromal Cell-Derived Factor-1α (SDF-1α; SDF) to increase the recruitment and infiltration of mesenchymal stem cells (MSCs) and Transforming Growth Factor-ß3 (TGF-ß3; TGF) to enhance cartilage tissue formation. To our knowledge, this study is the first to evaluate such a bioactive scaffold in a large animal model and demonstrates the capacity for dual growth factor release.

Resorbable Pins to Enhance Scaffold Retention in a Porcine Chondral Defect Model.

OBJECTIVE: Cartilage repair strategies have seen improvement in recent years, especially with the use of scaffolds that serve as a template for cartilage formation. However, current fixation strategies are inconsistent with regards to retention, may be technically challenging, or may damage adjacent tissues or the implant itself. Therefore, the goal of this study was to evaluate the retention and repair potential of cartilage scaffolds fixed with an easy-to-implement bioresorbable pin. DESIGN: Electrospun hyaluronic acid scaffolds were implanted into trochlear groove defects in 3 juvenile and 3 adult pigs to evaluate short-term retention (2 weeks; pin fixation vs. press-fit and fibrin fixation) and long-term repair (8 months; scaffold vs. microfracture), respectively. RESULTS: For the retention study, press-fit and fibrin fixation resulted in short-term scaffold dislodgment (n = 2 each), whereas pin fixation retained all scaffolds that were implanted (n = 6). Pin fixation did not cause any damage to the opposing patellar surface, and only minor changes in the subchondral bone were observed. For long-term repair, no differences were observed between microfracture and scaffold groups, in terms of second-look arthroscopy and indentation testing. On closer visualization with micro computed tomography and histology, a high degree of variability was observed between animals with regard to subchondral bone changes and cartilage repair quality, yet each Scaffold repair displayed similar properties to its matched microfracture control. CONCLUSIONS: In this study, pin fixation did not cause adverse events in either the short- or the long-term relative to controls, indicating that pin fixation successfully retained scaffolds within defects without inhibiting repair.

Transection of the medial meniscus anterior horn results in cartilage degeneration and meniscus remodeling in a large animal model.

The meniscus plays a central load-bearing role in the knee joint. Unfortunately, meniscus injury is common and can lead to joint degeneration and osteoarthritis (OA). In small animal models, progressive degenerative changes occur with the unloading of the meniscus via destabilization of the medial meniscus (DMM). However, few large animal models of DMM exist and the joint-wide initiation of the disease has not yet been defined in these models. Thus, the goal of this study is to develop and validate a large animal model of surgically induced DMM and to use multimodal (mechanical, histological, and magnetic resonance imaging) and multiscale (joint to tissue level) quantitative measures to evaluate degeneration in both the meniscus and cartilage. DMM was achieved using an arthroscopic approach in 13 Yucatan minipigs. One month after DMM, joint contact area decreased and peak pressure increased, indicating altered load transmission as a result of meniscus destabilization. By 3 months, the joint had adapted to the injury and load transmission patterns were restored to baseline, likely due to the formation and maturation of a fibrovascular scar at the anterior aspect of the meniscus. Despite this, we found a decrease in the indentation modulus of the tibial cartilage and an increase in cartilage histopathology scores at 1 month compared to sham-operated animals; these deleterious changes persisted through 3 months. Over this same time course, meniscus remodeling was evident through decreased proteoglycan staining in DMM compared to sham menisci at both 1 and 3 months. These findings support that arthroscopic DMM results in joint degeneration in the Yucatan minipig and provide a new large animal testbed in which to evaluate therapeutics and interventions to treat post-traumatic OA that originates from a meniscal injury.

GAPDH Overexpression in the T Cell Lineage Promotes Angioimmunoblastic T Cell Lymphoma through an NF-κB-Dependent Mechanism.

GAPDH is emerging as a key player in T cell development and function. To investigate the role of GAPDH in T cells, we generated a transgenic mouse model overexpressing GAPDH in the T cell lineage. Aged mice developed a peripheral Tfh-like lymphoma that recapitulated key molecular, pathological, and immunophenotypic features of human angioimmunoblastic T cell lymphoma (AITL). GAPDH induced non-canonical NF-κB pathway activation in mouse T cells, which was strongly activated in human AITL. We developed a NIK inhibitor to reveal that targeting the NF-κB pathway prolonged AITL-bearing mouse survival alone and in combination with anti-PD-1. These findings suggest the therapeutic potential of targeting NF-κB signaling in AITL and provide a model for future AITL therapeutic investigations.

Emerging therapies for cartilage regeneration in currently excluded 'red knee' populations.

The field of articular cartilage repair has made significant advances in recent decades; yet current therapies are generally not evaluated or tested, at the time of pivotal trial, in patients with a variety of common comorbidities. To that end, we systematically reviewed cartilage repair clinical trials to identify common exclusion criteria and reviewed the literature to identify emerging regenerative approaches that are poised to overcome these current exclusion criteria. The term "knee cartilage repair" was searched on clinicaltrials.gov. Of the 60 trials identified on initial search, 33 were further examined to extract exclusion criteria. Criteria excluded by more than half of the trials were identified in order to focus discussion on emerging regenerative strategies that might address these concerns. These criteria included age (<18 or >55 years old), small defects (<1 cm2), large defects (>8 cm2), multiple defect (>2 lesions), BMI >35, meniscectomy (>50%), bilateral knee pathology, ligamentous instability, arthritis, malalignment, prior repair, kissing lesions, neurologic disease of lower extremities, inflammation, infection, endocrine or metabolic disease, drug or alcohol abuse, pregnancy, and history of cancer. Finally, we describe emerging tissue engineering and regenerative approaches that might foster cartilage repair in these challenging environments. The identified criteria exclude a majority of the affected population from treatment, and thus greater focus must be placed on these emerging cartilage regeneration techniques to treat patients with the challenging "red knee".

Both cetaceans in the Brazilian Amazon show sustained, profound population declines over two decades.

Obligate river dolphins occur only in the rivers of Asia and South America, where they are increasingly subject to damaging pressures such as habitat degradation, food competition and entanglement in fishing gear as human populations expand. The Amazon basin hosts two, very different, dolphins-the boto or Amazon river dolphin (Inia geoffrensis) and the smaller tucuxi (Sotalia fluviatilis). Both species have wide geographical ranges and were once considered to be relatively abundant. Their IUCN Red List conservation status of Data Deficient (DD), due to limited information on threats, ecology, population numbers and trends, did not initially cause alarm. However, the development of dolphin hunting to provide fish bait at around the beginning of this millennium broadly coincided with the onset of a widespread perception that numbers of both species were in decline. Consequently, the need for population trend data to inform conservation advice and measures became urgent. This paper presents a 22-year time series of standardised surveys for both dolphins within the Mamirauá Reserve, Amazonas State, Brazil. Analysis of these data show that both species are in steep decline, with their populations halving every 10 years (botos) and 9 years (tucuxis) at current rates. These results are consistent with published, independent information on survival rates of botos in this area, which demonstrated a substantial drop in annual survival, commencing at around the year 2000. Mamirauá is a protected area, and is subject to fewer environmental pressures than elsewhere in the region, so there is no reason to suspect that the decline in dolphins within the Reserve is more pronounced than outside it. If South America's freshwater cetaceans are to avoid following their Asian counterparts on the path to a perilous conservation status, effective conservation measures are required immediately. Enforcement of existing fishery laws would greatly assist in achieving this.

Development of highly sensitive fluorescent probes for the detection of ß-galactosidase activity - application to the real-time monitoring of senescence in live cells.

We report the development of four novel fluorescent probes to monitor the activity of the ß-galactosidase enzyme (ß-gal), in vitro and in living cells. The fluorophores are based on a 6-amino-styryl-benzothiazole push-pull core and display a strong ICT emission. The probes encompass the fluorescent motif that is connected to a ß-d-galactopyranoside moiety through a self-immolative benzyl carbamate linker (ßGal-1-4). The screening of four different fluorophores enabled us to access new light-up and two-band ratiometric reporters. The four probes, ßGal-1-4, exhibited an extremely fast response and over 200-fold fluorescence enhancement (ßGal-1) following the enzymatic cleavage of the ß-d-galactopyranoside unit. This rapid and extremely sensitive response allowed the detection of senescence-associated ß-galactosidase (SA-ß-gal) activity; a widely used biomarker of senescence. More importantly, ßGal-1 also enabled us to monitor, in real-time, the emergence of senescence in live cells, i.e. the phenotypic transformation from normal to senescent cell. These findings underpin the fact that ßGal-1 may find useful applications in biomedical research. Importantly, ßGal-1 is suitable for epifluorescence and confocal microscopies, and flow cytometry techniques, which are among the most common analytical tools in biology.

Acute Radial Nerve Repair with Humeral Shaft Shortening and Fixation Following a Closed Humeral Shaft Fracture: A Case Report.

CASE: A 65-year-old woman sustained a closed segmental humeral shaft fracture with a complete radial nerve palsy. Radial nerve neurotmesis was found during acute surgical exploration and fixation. Treatment included a 2-cm shortening osteotomy of the humeral shaft and osteosynthesis in order to obtain a tension-free primary end-to-end repair of the radial nerve. CONCLUSION: Tension-free primary nerve repair can be technically challenging with humeral shaft fractures. A shortening osteotomy of the humeral shaft and osteosynthesis are feasible to achieve tension-free neurorrhaphy without nerve-grafting.

Assessment of new triplet forming artificial nucleobases as RNA ligands directed towards HCV IRES IIId loop.

We report the synthesis of two new artificial nucleobase scaffolds, 1 and 2, featuring adequate hydrogen bonding donors and acceptors for the molecular recognition of U:A and C:G base pairs, respectively. The tethering of these structures to various amino acids and the assessment of these artificial nucleobase-amino acid conjugates as RNA ligands against a model of HCV IRES IIId domain are also reported. Compound 1e displayed the highest affinity (Kd twice lower than neomycin - control). Moreover, it appears that this interaction is enthalpically and entropically favored.

Morphology and Ultrastructure of the Amazon River Dolphin (Inia geoffrensis) Spermatozoa.

The spermatozoa from seven adult Amazon river dolphins (Inia geoffrensis, CETACEA: INIIDAE) were analyzed by light and electron microscopy. The spermatozoa showed an elongated ellipsoid shaped head and a long tail with a well distinguishable midpiece. The head spermatozoa have a smooth surface like other odontocetes examined, with the exception of the Delphinidae family. The mean dimensions of the spermatozoa were within the range already reported for other cetaceans. The spermatozoa midpiece, as in other cetaceans, showed a random pattern of mitochondria, different from that described for other mammals. Further studies of sperm morphology of a wider spectrum of cetacean families could help to better understand the reproductive biology of these animals and the intergeneric and intrageneric relationships among them, as well as, among other mammals. Anat Rec, 300:1519-1523, 2017. © 2017 Wiley Periodicals, Inc.

In Vitro and in Vivo Evaluation of Fully Substituted (5-(3-Ethoxy-3-oxopropynyl)-4-(ethoxycarbonyl)-1,2,3-triazolyl-glycosides as Original Nucleoside Analogues to Circumvent Resistance in Myeloid Malignancies.

A series of nucleoside analogues bearing a 1,4,5-trisubstituted-1,2,3-triazole aglycone was synthesized using a straightforward click/electrophilic addition or click/oxidative coupling tandem procedures. SAR analysis, using cell culture assays, led to the discovery of a series of compounds belonging to the 5-alkynyl-1,2,3-triazole family that exhibits potent antileukemic effects on several hematologic malignancies including chronic myeloid leukemia (CML) and myelodysplastic syndromes (MDS) either sensitive or resistant to their respective therapy. Compound 4a also proved efficient in vivo on mice xenografted with SKM1-R MDS cell line. Additionally, some insights in its mode of action revealed that this compound induced cell death by caspase and autophagy induction.