Murine cancer cachexia models replicate elevated catabolic pembrolizumab clearance in humans.

BACKGROUND: Monoclonal antibody (mAb) immune checkpoint inhibitor (ICI) therapies have dramatically impacted oncology this past decade. However, only about one-third of patients respond to treatment, and biomarkers to predict responders are lacking. Recent ICI clinical pharmacology data demonstrate high baseline drug clearance (CL0) significantly associates with shorter overall survival, independent of ICI exposure, in patients receiving ICI mAb therapies. This suggests CL0 may predict outcomes from ICI therapy, and cachectic signalling may link elevated CL0 and poor response. Our aim was to determine if mouse models of cancer cachexia will be useful for studying these phenomena and their underlying mechanisms. METHODS: We evaluated pembrolizumab CL in the C26 and Lewis lung carcinoma mouse models of cancer cachexia. A single treatment of vehicle or pembrolizumab, at a dose of 2 or 10 mg/kg, was administered intravenously by tail vein injection. Pembrolizumab was quantified by an ELISA in serial plasma samples, and FcRn gene (Fcgrt) expression was assessed in liver using real-time quantitative reverse transcription PCR. Non-compartmental and mixed-effects pharmacokinetics analyses were performed. RESULTS: We observed higher pembrolizumab CL0 and decreased Fcgrt expression in whole liver tissue from tumour-bearing vs. tumour-free mice. In multivariate analysis, presence of tumour, total murine IgG, muscle weight and Fcgrt expression were significant covariates on CL, and total murine IgG was a significant covariate on V1 and Q. CONCLUSIONS: These data demonstrate increases in catabolic clearance of monoclonal antibodies observed in humans can be replicated in cachectic mice, in which Fcgrt expression is also reduced. Notably, FcRn activity is essential for proper antigen presentation and antitumour immunity, which may permit the study of cachexia's impact on FcRn-mediated clearance and efficacy of ICI therapies.

A Systematic Review of Intra-pancreatic Fat Deposition and Pancreatic Carcinogenesis.

BACKGROUND: Excess adiposity is considered causally related to pancreatic cancer. While most knowledge on the topic comes from studies on general and visceral adiposity, the role of intra-pancreatic fat deposition in pancreatic carcinogenesis just begins to be elucidated. The aim was to conduct a comprehensive systematic review of clinical studies on intra-pancreatic fat deposition in individuals with pancreatic cancer or pre-malignant lesions. METHODS: A literature search was conducted independently by two reviewers using three electronic databases. Studies were included if they reported on intra-pancreatic fat deposition determined based on modern radiology or histology. Summary estimates were presented as pooled prevalence or relative risk and 95% confidence interval. RESULTS: A total of 13 studies (encompassing 2178 individuals) were included. The pooled prevalence of intra-pancreatic fat deposition in individuals with pancreatic cancer or pre-malignant lesions was 52% (95% confidence interval, 38-66%). The presence of pancreatic cancer or pre-malignant lesions was associated with a significantly increased risk of intra-pancreatic fat deposition (relative risk 2.78 (95% confidence interval, 1.56-4.94, p < 0.001). CONCLUSION: Individuals with pancreatic cancer or pre-malignant lesions are characterized by increased intra-pancreatic fat deposition. There are sound grounds for conceptually viewing intra-pancreatic fat deposition as a combination of fat accumulation in the pancreas (due to expansion of excess visceral fat) and fatty replacement of the pancreas (due to changes in cellular identity within the pancreas). Guidelines on reporting intra-pancreatic fat deposition need to be developed with a view to informing a comprehensive and standardized characterization of this clinical entity in future studies.

Multiscale analysis of architecture, cell size and the cell cortex reveals cortical F-actin density and composition are major contributors to mechanical properties during convergent extension.

The large-scale movements that construct complex three-dimensional tissues during development are governed by universal physical principles. Fine-grained control of both mechanical properties and force production is crucial to the successful placement of tissues and shaping of organs. Embryos of the frog Xenopus laevis provide a dramatic example of these physical processes, as dorsal tissues increase in Young's modulus by six-fold to 80 Pascal over 8 h as germ layers and the central nervous system are formed. These physical changes coincide with emergence of complex anatomical structures, rounds of cell division, and cytoskeletal remodeling. To understand the contribution of these diverse structures, we adopt the cellular solids model to relate bulk stiffness of a solid foam to the unit size of individual cells, their microstructural organization, and their material properties. Our results indicate that large-scale tissue architecture and cell size are not likely to influence the bulk mechanical properties of early embryonic or progenitor tissues but that F-actin cortical density and composition of the F-actin cortex play major roles in regulating the physical mechanics of embryonic multicellular tissues.

Spatiotemporally Controlled Mechanical Cues Drive Progenitor Mesenchymal-to-Epithelial Transition Enabling Proper Heart Formation and Function.

During early cardiogenesis, bilateral fields of mesenchymal heart progenitor cells (HPCs) move from the anterior lateral plate mesoderm to the ventral midline, undergoing a mesenchymal-to-epithelial transition (MET) en route to forming a single epithelial sheet. Through tracking of tissue-level deformations in the heart-forming region (HFR) as well as movement trajectories and traction generation of individual HPCs, we find that the onset of MET correlates with a peak in mechanical stress within the HFR and changes in HPC migratory behaviors. Small-molecule inhibitors targeting actomyosin contractility reveal a temporally specific requirement of bulk tissue compliance to regulate heart development and MET. Targeting mutant constructs to modulate contractility and compliance in the underlying endoderm, we find that MET in HPCs can be accelerated in response to microenvironmental stiffening and can be inhibited by softening. To test whether MET in HPCs was responsive to purely physical mechanical cues, we mimicked a high-stress state by injecting an inert oil droplet to generate high strain in the HFR, demonstrating that exogenously applied stress was sufficient to drive MET. MET-induced defects in anatomy result in defined functional lesions in the larval heart, implicating mechanical signaling and MET in the etiology of congenital heart defects. From this integrated analysis of HPC polarity and mechanics, we propose that normal heart development requires bilateral HPCs to undergo a critical behavioral and phenotypic transition on their way to the ventral midline, and that this transition is driven in response to the changing mechanical properties of their endoderm substrate.

Evaluation of methanogenic activity of biogas plant slurry on ossein factory wastes.

The objective of the present work was to evaluate the ossein factory wastes, which include primary clarified bone waste (PCBW) and sinews for methane production, by monitoring methanogenic activity of predigested biogas plant slurry. A specific methanogenic activity of biogas plant slurry (anaerobic seed) was measured at 38 degrees C using different proportions of ossein factory wastes in an assay medium. The pH of slurry was intensively maintained until course of digestion. A moderate proportion of both substrates showed a maximum methane production at 20 days of incubation in batch mode. However, a maximum cumulative methane yield achieved by biogas plant slurry on PCBW was low as compared to sinews. The best organic matter degradation was achieved even at a high proportion of ossein factory wastes used in digesters. These substitutes would be useful, without seriously reducing total gas production, for methane production if they partially mixed with cattle dung. As a result of this preliminary study, we suggest that ossein factory wastes are potential alternative sources for biogas production in ossein factory.

Evaluation of methanogenic activity of biogas plant slurry for monitoring codigestion of ossein factory wastes and cyanobacterial biomass.

Overall measurement of methanogenic activity of sludge and or slurry is thought as a key for understanding the basic physiology of anaerobic consortia involved in anaerobic digestion process of an alternative biomass. In this study, the methanogenic activity of biogas plant slurry was used to evaluate the anaerobic digestion of ossein factory wastes such as sinews and primary clarified bone waste (PCBW) and cyanobacterial biomass in standard assay conditions. A maximum methanogenic activity was reported here when ossein factory wastes mixed with cyanobacterial biomass in specific proportions in which sinews and PCBW alone also favored to a significant methane yield. Cyanobacterial biomass alone did not give a desirable methanogenic activity. Approximately 48% of total solids were destroyed from these wastes after 30 days. This study gives information on the use of these wastes with suitable proportions for taking an effort in a large-scale anaerobic digestion in an effective way of ossein factory.

Biodiversity of epilithic cyanobacteria from freshwater streams of Kakoijana reserve forest, Assam, India.

The biodiversity of epilithic cyanobacteria from one of the unexplored habitats of freshwater streams of Kakoijana reserve forest of Assam, India was estimated. This paper lists a total of 29 species representing 18 genera of 12 families and 4 orders as per recent system of classification. Morphological descriptions, common habitats and distribution pattern were described for each species identified that were represented systematically. Of these 29 species, 11 were unicellular, 9 non-heterocytous filamentous and 9 heterocytous filamentous forms. All the unicellular (Aphanocapsa crassa, A. muscicola, Aphanothece nidulans, A. saxicola, Chlorogloea purpurea, Chroococcus cohaerens, C. minimus, C. minor, Cyanobacterium cedrorum, Cyanocystis versicolor and Gloeocapsopsis crepidinum) and 13 (Calothrix epiphytica, C. scopulorum, Leptolyngbya boryana, L. calotrichoides, L. fragilis, L. notata, Lyngbya arboricola, Nostoc humifusum, N. oryzae, N. punctiforme, Parthasarathiella prolifica, Porphyrosiphon ceylanicus and Scytonema millei) of the remaining 18 species were recorded for the first time as freshwater epiliths. While, 5 species (Hapalosiphon welwitschii, Leptolyngbya tenuis, Oscillatoria pseudogeminata, Phormidium laetevirens, Tolypothrix fragilis) and 8 species (Aphanothece saxicola, Calothrix scopulorum Chlorogloea purpurea, Chroococcus minor, Gloeocapsopsis crepidinum, Leptolyngbya calotrichoides, L. fragilis and L. tenuis) were reported earlier as freshwater-and marine-epilithic forms respectively. All are new records for Assam except 6 species (A. nidulans, H. welwitschii, N. punctiforme, N. oryzae, O. pseudogeminata and P. ceylanicus), while 3 species (C. purpurea, L. boryana and L. calotrichoides) are new records for India. Six nitrogen fixing heterocytous forms such as, C. epiphytica, C. scopulorum, N. humifusum, N. punctiforme, N. oryzae and S. millei, were common to the neighboring paddy fields.

Modified helix-loop-helix motifs of calmodulin--The influence of the exchange of helical regions on calcium-binding affinity.

The four calcium-binding sites, called the helix-loop-helix, or the EF-hand motifs, of calmodulin differ in their ion-binding affinities; this has been thought to arise due to the variations in the sequences of the loop regions where the ion binds. We focus attention here on the role of the flanking helical regions on the calcium-binding affinities. Peptides were synthesized in a manner that simulates the E and F helical flanks of site 4 (the strongest calcium-binding site of the calmodulin) to sandwich the loop sequences of sites 1, 2, 3 and 4 so as to produce peptides named 414, 424, 434 and 444, as well as using the helical flanks of site 1 (the weakest site) to produce peptides 111, 121, 131 and 141. Calcium binding was monitored using the calcium-mimic dye Stains-all (4,4,4',5'-dibenzo-3,3'-diethyl-9-methyl-thiacarbocyanine bromide). Binding abilities were seen to increase several-fold when the E and F helices of site 1 were replaced by those of site 4 (i.e., 111-414). In contrast, the intensity of circular dichroism induced in the absorption bands of the bound achiral dye decreased significantly when the helical flanks of site 4 were replaced with those of site 1 (i.e., 444-141). The helical flanks of site 4 impart greater binding ability to a given loop region, while the helical flanks of site 1 tend to weaken it.

A conformational study of corneal dermatan sulfate proteoglycan using fluorescence spectroscopy.

DSPG is a major proteoglycan of the corneal stroma and is thought to be important for the transparency of the tissue. We have studied its conformation by exploring the microenvironment and dynamics of its lone Tryptophan (Trp) residue using steady state and time resolved fluorescence spectroscopy. DSPG exhibits a doublet Trp fluorescence emission. Such a doublet emission has been observed earlier in the copper protein azurin and in avian lens delta-crystallin. Unlike the above cases where the doublet emission is thought to arise due to vibronic structure or the location of Trp at the interface of interacting subunits, fluorescence quenching, denaturation studies and ANS binding with DSPG indicate the location of Trp at two different environments. Such a situation could arise from the differential glycosylation of the core protein or due to duplexation and aggregation of the glycosaminoglycan chains.

Effect of UVB radiation on corneal aldehyde dehydrogenase.

PURPOSE: A Class 3 aldehyde dehydrogenase happens to be a major soluble protein constituent of the cornea. Its role is conjectured to be manifold: to protect the tissue from oxidative damage by eliminating the toxic aldehydes produced upon lipid peroxidation under oxidative stress, to act as an UV-absorber, and to maintain the level of the coenzyme NADH in the cornea. We have studied the effect of UVB on the structure and enzyme activity of corneal aldehyde dehydrogenase. METHODS: Aldehyde dehydrogenase was irradiated at 295 nm for varying periods of time and change in its enzyme activity assayed. The structural changes in the molecule accompanying irradiation were monitored using fluorescence and circular dichroism spectroscopy, and its hydrodynamic behavior and surface hydrophobicity studied using gel filtration chromatography and binding of the hydrophobic fluorophore ANS. The protective ability of aldehyde dehydrogenase in preventing aggregation of photolabile proteins, such as Gamma-crystallin of the eye lens, was studied by monitoring the scattering value of the test protein with irradiation by UVB. RESULTS: Aldehyde dehydrogenase is seen to undergo photodamage with alterations in its quaternary structure, though no significant change is noticed in the peptide chain conformation. Under such conditions the molecule continues to act as a protectant by preventing aggregation of photolabile proteins such as the eye lens Gamma-crystallin. CONCLUSIONS: Our earlier studies have shown that the free sulfhydryl groups are important for the antioxidant abilities of aldehyde dehydrogenase. Its protective ability towards photoaggregation of Gamma-crystallin seen here might arise both due to: (i) oxyradical quenching and (ii) the increased surface hydrophobicity of the molecule upon irradiation, which allows it to bind to, and thus inhibit the aggregation of interacting proteins.

Conformation, stability and interactions of corneal keratan sulfate proteoglycan.

We have monitored the molecular conformation, stability, interaction and dynamics of keratan sulfate proteoglycan, the major structural protein component of the cornea, in solution, by studying the fluorescence spectral features of its tryptophan residues as component-specific intrinsic spectral probes (collagen, the other structural component of the cornea, has no tryptophans). Our study suggests that the Trp region of the molecule is in a motionally restricted environment as it exhibits a fluorescence red-edge effect and shows dipole relaxation. The extrinsic spectral probe 8-anilinonaphthalene 1-sulfonate reveals keratan sulfate proteoglycan to possess significant surface hydrophobicity. This dual character of keratan sulfate proteoglycan allows us to label it as an 'ambidextran' proteoglycan. The molecule is stable between pH 5-8 and has a Tm value of 72 degrees C. Disulfide bonds play a role in the stability of the molecule. KSPG is seen to interact with collagen and the model compound, poly(L-proline). Interaction of the proteoglycan with unilamellar vesicles appears to be more interfacial than penetrative. This dual interaction displayed by KSPG with collagen and with lipid assemblages suggests that it plays the role of a 'filler' in the extracellular matrix of the cornea.

Fluorescence properties of isolated intact normal human corneas.

We have examined the fluorescence properties of excised intact normal human corneas from over a hundred donors, using synchronous excitation fluorescence spectroscopy. In some of the corneas from the donors, a fluorophore with an excitation band centered at 330 nm was observed. This fluorophore does not seem to correspond to the dityrosine moiety or to any photoproducts of tryptophan. Isolated corneas irradiated with light of 295 nm wavelength do not produce any fluorescent photoproducts, suggesting that the intact tissue has endogenous quenchers, radical scavengers and antioxidants that inhibit its photodamage. The non-tryptophan fluorophores that accumulate in some corneas thus appear to arise largely from the nonenzymatic glycosylation (glycation) of the constituent proteins as similar fluorophores are detected in the corneas of rats in which diabetes is induced.

In situ fluorescence spectroscopic studies on bovine cornea.

The cornea is a transparent ocular tissue and its transparency is thought to be a result of intramolecular interactions and the supramolecular organization of its protein constituents. We have studied the intrinsic fluorescence properties of intact bovine corneas and compared these with that of the opaque sclera. It was observed that with increasing excitation wavelengths the emission maxima shifted toward the red edge exhibiting the phenomenon of red edge excitation shift, which is indicative of immobilization of the constituent fluorophores. The magnitude of the shift increased after photodamage by irradiation at 295 nm. Many of the spectral characteristics of the cornea are shown to be due to its proteoglycans, which show surprisingly significant red edge excitation shift in solution.