Relative Bioavailability Assessment of Solid Forms by An Artificial Stomach and Duodenum Apparatus.

In this work, the ability of the artificial stomach and duodenum (ASD) model to predict bioavailability in rats was investigated using a poorly soluble model compound, BI-639667. A solution and four suspensions of different solid forms of BI-639667 were tested both in an ASD and rats. Rank order of the bioavailability estimated from an ASD apparatus is consistent with that of in vivo result in rats, i.e., solution > salicylic acid cocrystal > malate salt > maleate salt > monohydrate, which correlates with the ability of the different solid forms to maintain supersaturation with respect to the stable form in aqueous solution. The results support the use of an ASD for characterizing dissolution performance of solid forms to aid their selection for tablet formulation development.

What's in a Name? Replacing Pejorative Terminology in Anatomic Pathology.

Recent changes to medical terminology and nomenclature have made strides to improve ethical integrity in healthcare. Removing tarnished eponymous namesakes and depreciative descriptions from the medical lexicon is a challenging, but righteous undertaking. In this article, the authors propose replacing the pejorative histologic description of "Indian file" with "Tusken Raider file."

Human Serum Albumin Immobilized On Magnetizable Beads: A Rapid Method for Compound HSA Binding Study.

Immobilized human serum albumin (HSA) was developed by coupling His-tagged HSA onto Ni2+-coupled magnetizable beads (HSA-beads), allowing the HSA to be easily removed from incubation components. The HSA-beads system provides a rapid and convenient method to study HSA compound binding. In this study, the HSA-beads system was characterized and evaluated as a tool for assessing compound HSA binding properties. The free fraction (fu) values of test compounds measured using HSA-beads were comparable to those determined by equilibrium dialysis (ED), which is commonly used to evaluate albumin binding in vitro. The equilibrium dissociation constant (Kd) values determined for a series of compounds using the HSA-beads method demonstrated good correlation with literature data. This good correlation also suggests that the binding of His-HSA to the beads does not impact the conformations of the two compound binding sites of HSA, as the range of compounds tested encompassed binding to both sites. Furthermore, the Kd values of representative compounds itraconazole and BIRT2584 that were difficult to assess using ED, due to significant cellulose membrane adsorption, were successfully determined. The HSA-beads provide several advantages over ED, such as simple preparation, short assay incubation duration, and the ability to quantify both free and HSA-bound species of the test compound, facilitated by the simple separation of HSA-beads from the solution phase using a magnetic field. These properties render the HSA-beads method suitable for high-throughput studies on compound HSA binding.

The morphology, functionality, and longevity of a novel all human hepatic cell-based tri-culture system.

There remains a significant need for a convenient, phenotypically stable long-term culture platform for primary human hepatocytes (PHHs) for use in pharmacological and toxicological applications. Conventional in vitro models are often inconvenient, burdensome to use, and unable to support a multitude of donor lots or maintain PHH structural and functional properties over extended time. To address these limitations, an all-human cell-based hepatic tri-culture system (HTCS) has been developed comprised of frozen vials of PHHs and feeder cells. Qualified PHHs exhibited healthy morphological characteristics for ≥30 days. Extensive anastomosing networks of bile canaliculi with tight and gap junctions were established early and remained stable and functional throughout the culture period. After 5 culture days, albumin, urea, and basal Phase 1 and Phase 2 metabolic functions were stable for at least 2 weeks and significantly higher in the HTCS PHHs compared to sandwich monoculture PHHs. Induction of CYP functional activity by prototypical receptor agonists was stable after 4 days for at least 2 weeks. Gene expression of Alb and various CYPs in the HTCS PHHs was significantly higher compared to sandwich monoculture PHHs. The HTCS represents a convenient, phenotypically stable, all-human PHH culture platform for pharmacological and toxicological applications.

Facing death, returning to life: A qualitative analysis of MDMA-assisted therapy for anxiety associated with life-threatening illness.

Anxiety associated with life-threatening illness (LTI) is a pervasive mental health issue with a wide impact. A spectrum of traditional pharmacotherapies and psychotherapies are available, but offer varying success in reducing symptoms and improving quality of life. We explore a novel therapy for this condition by assessing prominent thematic elements from participant narrative accounts of a pilot phase 2 clinical trial of 3,4 Methylenedioxymethamphetamine-Assisted Therapy (MDMA-AT) for treating anxiety associated with LTI. Semi-structured qualitative interviews were conducted with a subset of adult participants 3 months following completion of this trial. This qualitative analysis sought to complement, clarify, and expand upon the quantitative findings obtained from the clinical trial to further understand the process and outcomes of the treatment. Interviews were coded and analyzed using an Interpretative Phenomenological Analysis (IPA) methodological framework. Participants described in detail their experiences from before, during and after the trial, which were analyzed and categorized into thematic clusters. Specifically, participants explored what they felt were important elements of the therapeutic process including processing trauma and grief, exploring mystical and existential experiences, engaging with the present moment with reduced physiological activation, and facing illness and existential fears. Outcomes of the treatment included increased ability to cope with LTI, reduced psychological symptoms, improved vitality and quality of life, and feeling more resourced. Participant narratives also showed a reconnection to life and greater emotional resilience in response to trauma and medical relapse. These findings are compared to similar treatments for the same indication. Limitations and challenges encountered in conducting this study are discussed along with implications for theory and clinical treatment.

Shock wave formation from head-on collision of two subsonic vortex rings.

Vortex ring collisions have attracted intense interest in both water and air studies (Baird in Proc R Soc Lond Ser Math Phys Sci 409:59-65, 1987, Poudel et al. in Phys Fluids 33:096105, 2021, Lim and Nickels in Nature 357:225, 1992, New et al. in Exp Fluids 57:109, 2016, Suzuki et al. in Geophys Res Lett 34, 2007, Yan et al. in J Fluids Eng 140:054502, 2018, New et al. in J Fluid Mech 899, 2020, Cheng et al. in Phys Fluids 31:067107, 2019, Hernández and Reyes in 29:103604, 2017, Mishra et al. in Phys Rev Fluids, 2021, Zednikova et al. in Chem Eng Technol 42:843-850, 2019, Kwon et al. in Nature 600:64-69, 2021). These toroidal structures spin around a central axis and travel in the original direction of impulse while spinning around the core until inertial forces become predominant causing the vortex flow to spontaneously decay to turbulence (Vortex Rings, https://projects.iq.harvard.edu/smrlab/vortex-rings ). Previous studies have shown the collision of subsonic vortex rings resulting in reconnected vortex rings, but the production of a shock wave from the collision has not been demonstrated visibly (Lim and Nickels in Nature 357:225, 1992, Cheng et al. in Phys Fluids 31:067107, 2019). Here we present the formation of a shock wave due to the collision of explosively formed subsonic vortex rings. As the vortex rings travel at Mach 0.66 toward the collision point, they begin to trap high pressure air between them. Upon collision, high pressure air was imploded and released radially away from the axis of the collision, generating a visible shock wave traveling through and away from the colliding vortices at Mach 1.22. Our results demonstrate a pressure gradient with high pressure release creating a shock wave. We anticipate our study to be a starting point for more explosively formed vortex collisions. For example, explosives with different velocities of detonation could be tested to produce vortex rings of varying velocities.

Does large dam removal restore downstream riparian vegetation diversity? Testing predictions on the Elwha River, Washington, USA.

Large dams and their removal can profoundly affect riparian ecosystems by altering flow and sediment regimes, hydrochory, and landform dynamics, yet few studies have documented these effects on downstream plant communities. Ecological theory and empirical results suggest that by altering disturbance regimes, reducing hydrochory, and shifting communities to later successional stages, dams reduce downstream plant diversity. Dam removal could reverse these processes, but the release of large volumes of sediment could have unexpected, transient effects. Two large dams were removed on the Elwha River in Washington State, USA, from 2011 to 2014, representing an unprecedented opportunity to study large dam removal effects on riparian plant communities. Our research objectives were to determine: (1) whether the Elwha River dams were associated with lower downstream plant diversity and altered species composition across riparian landforms pre-dam removal, and (2) whether dam removal has begun to restore downstream diversity and composition. To address these objectives, we compared plant species richness and community composition in river segments above, below, and between the two dams. Plant communities were sampled twice before (2005 and 2010) and four times after (2013, 2014, 2016, and 2017) the start of dam removal, with 2013 and 2014 sampled while the upstream dam removal was ongoing. Prior to dam removal, native species richness was 41% lower below dams compared with the upstream segment; 6 years after dam removal began, it increased ~31% between the dams, whereas nonnative species richness and cover were not apparently affected by dams or their removal. Deposition caused by large volumes of released reservoir sediment had mixed effects on native species richness (increased on floodplains, decreased elsewhere) in the lowest river segment. Plant community composition was also different downstream from dams compared with the upstream reference, and has changed in downstream floodplains and bars since dam removal. In the long term, we expect that diversity will continue to increase in downstream river segments. Our results provide evidence that (1) large dams reduce downstream native plant diversity, (2) dam removal may restore it, and (3) given the natural dynamics of riparian vegetation, long-term, multiyear before-and-after monitoring is essential for understanding dam removal effects.

Shotgun Proteomics Sample Processing Automated by an Open-Source Lab Robot.

Mass spectrometry-based shotgun proteomics experiments require multiple sample preparation steps, including enzymatic protein digestion and clean-up, which can take up significant person-hours of bench labor and present a source of batch-to-batch variability. Lab automation with pipetting robots can reduce manual work, maximize throughput, and increase research reproducibility. Still, the steep starting prices of standard automation stations make them unaffordable for many academic laboratories. This article describes a proteomics sample preparation workflow using an affordable, open-source automation system (The Opentrons OT-2), including instructions for setting up semi-automated protein reduction, alkylation, digestion, and clean-up steps; as well as accompanying open-source Python scripts to program the OT-2 system through its application programming interface.

New fossils of Australopithecus sediba reveal a nearly complete lower back.

Adaptations of the lower back to bipedalism are frequently discussed but infrequently demonstrated in early fossil hominins. Newly discovered lumbar vertebrae contribute to a near-complete lower back of Malapa Hominin 2 (MH2), offering additional insights into posture and locomotion in Australopithecus sediba. We show that MH2 possessed a lower back consistent with lumbar lordosis and other adaptations to bipedalism, including an increase in the width of intervertebral articular facets from the upper to lower lumbar column ('pyramidal configuration'). These results contrast with some recent work on lordosis in fossil hominins, where MH2 was argued to demonstrate no appreciable lordosis ('hypolordosis') similar to Neandertals. Our three-dimensional geometric morphometric (3D GM) analyses show that MH2's nearly complete middle lumbar vertebra is human-like in overall shape but its vertebral body is somewhat intermediate in shape between modern humans and great apes. Additionally, it bears long, cranially and ventrally oriented costal (transverse) processes, implying powerful trunk musculature. We interpret this combination of features to indicate that A. sediba used its lower back in both bipedal and arboreal positional behaviors, as previously suggested based on multiple lines of evidence from other parts of the skeleton and reconstructed paleobiology of A. sediba.

One of the defining features of humans is our ability to walk comfortably on two legs. To achieve this, our skeletons have evolved certain physical characteristics. For example, the lower part of the human spine has a forward curve that supports an upright posture; whereas the lower backs of chimpanzees and other apes ­ which walk around on four limbs and spend much of their time in trees ­ lack this curvature. Studying the fossilized back bones of ancient human remains can help us to understand how we evolved these features, and whether our ancestors moved in a similar way. Australopithecus sediba was a close-relative of modern humans that lived about two million years ago. In 2008, fossils from an adult female were discovered at a cave site in South Africa called Malapa. However, the fossils of the lower back region were incomplete, so it was unclear whether the female ­ referred to as Malapa Hominin 2 (MH2) ­ had a forward-curving spine and other adaptations needed to walk on two legs. Here, Williams et al. report the discovery of new A. sediba fossils from Malapa. The new fossils are mainly bones from the lower back, and they fit together with the previously discovered MH2 fossils, providing a nearly complete lower spine. Analysis of the fossils suggested that MH2 would have had an upright posture and comfortably walked on two legs, and the curvature of their lower back was similar to modern females. However, other aspects of the bones' shape suggest that as well as walking, A. sediba probably spent a significant amount of time climbing in trees. The findings of Williams et al. provide new insights in to our evolutionary history, and ultimately, our place in the natural world around us. Our lower back is prone to injury and pain associated with posture, pregnancy and exercise (or lack thereof). Therefore, understanding how the lower back evolved may help us to learn how to prevent injuries and maintain a healthy back.

Transcriptome features of striated muscle aging and predictability of protein level changes.

We performed total RNA sequencing and multi-omics analysis comparing skeletal muscle and cardiac muscle in young adult (4 months) vs. early aging (20 months) mice to examine the molecular mechanisms of striated muscle aging. We observed that aging cardiac and skeletal muscles both invoke transcriptomic changes in innate immune system and mitochondria pathways but diverge in extracellular matrix processes. On an individual gene level, we identified 611 age-associated signatures in skeletal and cardiac muscles, including a number of myokine and cardiokine encoding genes. Because RNA and protein levels correlate only partially, we reason that differentially expressed transcripts that accurately reflect their protein counterparts will be more valuable proxies for proteomic changes and by extension physiological states. We applied a computational data analysis workflow to estimate which transcriptomic changes are more likely relevant to protein-level regulation using large proteogenomics data sets. We estimate about 48% of the aging-associated transcripts predict protein levels well (r ≥ 0.5). In parallel, a comparison of the identified aging-regulated genes with public human transcriptomics data showed that only 35-45% of the identified genes show an age-dependent expression in corresponding human tissues. Thus, integrating both RNA-protein correlation and human conservation across data sources, we nominate 134 prioritized aging striated muscle signatures that are predicted to correlate strongly with protein levels and that show age-dependent expression in humans. The results here reveal new details into how aging reshapes gene expression in striated muscles at the transcript and protein levels.

Homoplasy in the evolution of modern human-like joint proportions in Australopithecus afarensis.

The evolution of bipedalism and reduced reliance on arboreality in hominins resulted in larger lower limb joints relative to the joints of the upper limb. The pattern and timing of this transition, however, remains unresolved. Here, we find the limb joint proportions of Australopithecus afarensis, Homo erectus, and Homo naledi to resemble those of modern humans, whereas those of A. africanus, Australopithecus sediba, Paranthropus robustus, Paranthropus boisei, Homo habilis, and Homo floresiensis are more ape-like. The homology of limb joint proportions in A. afarensis and modern humans can only be explained by a series of evolutionary reversals irrespective of differing phylogenetic hypotheses. Thus, the independent evolution of modern human-like limb joint proportions in A. afarensis is a more parsimonious explanation. Overall, these results support an emerging perspective in hominin paleobiology that A. afarensis was the most terrestrially adapted australopith despite the importance of arboreality throughout much of early hominin evolution.

Characterization of ecotin homologs from Campylobacter rectus and Campylobacter showae.

Ecotin, first described in Escherichia coli, is a potent inhibitor of a broad range of serine proteases including those typically released by the innate immune system such as neutrophil elastase (NE). Here we describe the identification of ecotin orthologs in various Campylobacter species, including Campylobacter rectus and Campylobacter showae residing in the oral cavity and implicated in the development and progression of periodontal disease in humans. To investigate the function of these ecotins in vitro, the orthologs from C. rectus and C. showae were recombinantly expressed and purified from E. coli. Using CmeA degradation/protection assays, fluorescence resonance energy transfer and NE activity assays, we found that ecotins from C. rectus and C. showae inhibit NE, factor Xa and trypsin, but not the Campylobacter jejuni serine protease HtrA or its ortholog in E. coli, DegP. To further evaluate ecotin function in vivo, an E. coli ecotin-deficient mutant was complemented with the C. rectus and C. showae homologs. Using a neutrophil killing assay, we demonstrate that the low survival rate of the E. coli ecotin-deficient mutant can be rescued upon expression of ecotins from C. rectus and C. showae. In addition, the C. rectus and C. showae ecotins partially compensate for loss of N-glycosylation and increased protease susceptibility in the related pathogen, Campylobacter jejuni, thus implicating a similar role for these proteins in the native host to cope with the protease-rich environment of the oral cavity.

B-Cell Acute Lymphoblastic Leukemia Presenting as Leukemia Cutis: A Case Report.

Leukemia cutis (LC) is a manifestation of leukemia with infiltration of the dermis, epidermis, or subcutis by malignant leukocytes resulting in papules, plaques, nodules, or ulcers. It is usually associated with acute and chronic myeloid leukemia as well as T-cell acute lymphoblastic leukemia (T-ALL) but is very rare in patients with B-cell acute lymphoblastic leukemia (B-ALL). We report a case of a 58-year-old Hispanic male who presented with a non-healing leg ulcer of three months along with patches on the face, left arm, and bilateral legs with white blood cell (WBC) count of 50800/mm3 with 83% blasts, and flow cytometry findings of B-ALL. Punch biopsies from affected skin showed numerous dermal nodules composed of large atypical cells with open chromatin and prominent nucleoli. Immunohistochemical stains were consistent with B-ALL involving the skin and a diagnosis of LC was rendered. A high index of suspicion in relevant cases and prompt diagnosis is imperative to prevent any delays in appropriate therapy. Diagnosis in our case was aided by concurrent identification of B-ALL in the patient's peripheral blood. Since this information may not always be available, it is important to keep B-ALL in the differential any time there is a neoplastic infiltration of leukocytes in the dermis.

Proteomic signatures of acute oxidative stress response to paraquat in the mouse heart.

The heart is sensitive to oxidative damage but a global view on how the cardiac proteome responds to oxidative stressors has yet to fully emerge. Using quantitative tandem mass spectrometry, we assessed the effects of acute exposure of the oxidative stress inducer paraquat on protein expression in mouse hearts. We observed widespread protein expression changes in the paraquat-exposed heart especially in organelle-containing subcellular fractions. During cardiac response to acute oxidative stress, proteome changes are consistent with a rapid reduction of mitochondrial metabolism, coupled with activation of multiple antioxidant proteins, reduction of protein synthesis and remediation of proteostasis. In addition to differential expression, we saw evidence of spatial reorganizations of the cardiac proteome including the translocation of hexokinase 2 to more soluble fractions. Treatment with the antioxidants Tempol and MitoTEMPO reversed many proteomic signatures of paraquat but this reversal was incomplete. We also identified a number of proteins with unknown function in the heart to be triggered by paraquat, suggesting they may have functions in oxidative stress response. Surprisingly, protein expression changes in the heart correlate poorly with those in the lung, consistent with differential sensitivity or stress response in these two organs. The results and data set here could provide insights into oxidative stress responses in the heart and avail the search for new therapeutic targets.

Splice-Junction-Based Mapping of Alternative Isoforms in the Human Proteome.

The protein-level translational status and function of many alternative splicing events remain poorly understood. We use an RNA sequencing (RNA-seq)-guided proteomics method to identify protein alternative splicing isoforms in the human proteome by constructing tissue-specific protein databases that prioritize transcript splice junction pairs with high translational potential. Using the custom databases to reanalyze ∼80 million mass spectra in public proteomics datasets, we identify more than 1,500 noncanonical protein isoforms across 12 human tissues, including ∼400 sequences undocumented on TrEMBL and RefSeq databases. We apply the method to original quantitative mass spectrometry experiments and observe widespread isoform regulation during human induced pluripotent stem cell cardiomyocyte differentiation. On a proteome scale, alternative isoform regions overlap frequently with disordered sequences and post-translational modification sites, suggesting that alternative splicing may regulate protein function through modulating intrinsically disordered regions. The described approach may help elucidate functional consequences of alternative splicing and expand the scope of proteomics investigations in various systems.

The African ape-like foot of Ardipithecus ramidus and its implications for the origin of bipedalism.

The ancestral condition from which humans evolved is critical for understanding the adaptive origin of bipedal locomotion. The 4.4 million-year-old hominin partial skeleton attributed to Ardipithecus ramidus preserves a foot that purportedly shares morphometric affinities with monkeys, but this interpretation remains controversial. Here I show that the foot of Ar. ramidus is most similar to living chimpanzee and gorilla species among a large sample of anthropoid primates. The foot morphology of Ar. ramidus suggests that the evolutionary precursor of hominin bipedalism was African ape-like terrestrial quadrupedalism and climbing. The elongation of the midfoot and phalangeal reduction in Ar. ramidus relative to the African apes is consistent with hypotheses of increased propulsive capabilities associated with an early form of bipedalism. This study provides evidence that the modern human foot was derived from an ancestral form adapted to terrestrial plantigrade quadrupedalism.

Identifying High-Priority Proteins Across the Human Diseasome Using Semantic Similarity.

Identifying the genes and proteins associated with a biological process or disease is a central goal of the biomedical research enterprise. However, relatively few systematic approaches are available that provide objective evaluation of the genes or proteins known to be important to a research topic, and hence researchers often rely on subjective evaluation of domain experts and laborious manual literature review. Computational bibliometric analysis, in conjunction with text mining and data curation, attempts to automate this process and return prioritized proteins in any given research topic. We describe here a method to identify and rank protein-topic relationships by calculating the semantic similarity between a protein and a query term in the biomerical literature while adjusting for the impact and immediacy of associated research articles. We term the calculated metric the weighted copublication distance (WCD) and show that it compares well to related approaches in predicting benchmark protein lists in multiple biological processes. We used WCD to extract prioritized "popular proteins" across multiple cell types, subanatomical regions, and standardized vocabularies containing over 20 000 human disease terms. The collection of protein-disease associations across the resulting human "diseasome" supports data analytical workflows to perform reverse protein-to-disease queries and functional annotation of experimental protein lists. We envision that the described improvement to the popular proteins strategy will be useful for annotating protein lists and guiding method development efforts as well as generating new hypotheses on understudied disease proteins using bibliometric information.

A conserved DGGK motif is essential for the function of the PglB oligosaccharyltransferase from Campylobacter jejuni.

In Campylobacter jejuni, the PglB oligosaccharyltransferase catalyzes the transfer of a heptasaccharide from a lipid donor to asparagine within the D/E-X1-N-X2-S/T sequon (X1,2 ≠ P) or releases this heptasaccharide as free oligosaccharides (fOS). Using available crystal structures and sequence alignments, we identified a DGGK motif near the active site of PglB that is conserved among all Campylobacter species. We demonstrate that amino acid substitutions in the aspartate and lysine residues result in loss of protein glycosylation in the heterologous Escherichia coli system. Similarly, complementation of a C. jejuni pglB knock-out strain with mutated pglB alleles results in reduced levels of N-linked glycoproteins and fOS in the native host. Analysis of the PglB crystal structures from Campylobacter lari and the soluble C-terminal domain from C. jejuni suggests a particularly important structural role for the aspartate residue and the two following glycine residues, as well as a more subtle, less defined role for the lysine residue. Limited proteolysis experiments indicate that conformational changes of wildtype PglB that are induced by the binding of the lipid-linked oligosaccharide are altered by changes in the DGGK motif. Related to these findings, certain Campylobacter species possess two PglB orthologues and we demonstrate that only the orthologue containing the DGGK motif is active. Combining the knowledge gained from the PglB structures and mutagenesis studies, we propose a function for the DGGK motif in affecting the binding of the undecaprenyl-pyrophosphate glycan donor substrate that subsequently influences N-glycan and fOS production.

Disseminated Kaposi sarcoma with osseous metastases in an HIV-positive patient.

Kaposi sarcoma is a neoplasm commonly associated with human herpesvirus 8 and HIV/AIDS. We present a 44-year-old African immigrant woman who presented to the emergency department after several months of abdominal pain. She was found to be HIV positive, and computed tomography demonstrated numerous lesions of the lungs, liver, and spleen, gastric wall thickening, and several lytic lesions of the spine. Fluoroscopy-guided biopsy of a lytic lesion of the spine yielded the diagnosis of Kaposi sarcoma. AIDS-related Kaposi sarcoma with osseous involvement is rare, with approximately 30 cases reported in the literature. When osteolytic lesions are encountered in an HIV-positive patient, Kaposi sarcoma should remain in the differential.