Most multicellular organisms are freeze sensitive, but the ability to survive freezing of the extracellular fluids evolved in several vertebrate ectotherms, some plants, and many insects. Here, we test the coupled hypotheses that are perpetuated in the literature: that irreversible denaturation of proteins and loss of biological membrane integrity are two ultimate molecular mechanisms of freezing injury in freeze-sensitive insects and that seasonally accumulated small cryoprotective molecules (CPs) stabilize proteins and membranes against injury in freeze-tolerant insects. Using the drosophilid fly,Chymomyza costata, we show that seven different soluble enzymes exhibit no or only partial loss of activity upon lethal freezing stress applied in vivo to whole freeze-sensitive larvae. In contrast, the enzymes lost activity when extracted and frozen in vitro in a diluted buffer solution. This loss of activity was fully prevented by adding low concentrations of a wide array of different compounds to the buffer, includingC. costatanative CPs, other metabolites, bovine serum albumin (BSA), and even the biologically inert artificial compounds HistoDenz and Ficoll. Next, we show that fat body plasma membranes lose integrity when frozen in vivo in freeze-sensitive but not in freeze-tolerant larvae. Freezing fat body cells in vitro, however, resulted in loss of membrane integrity in both freeze-sensitive and freeze-tolerant larvae. Different additives showed widely different capacities to protect membrane integrity when added to in vitro freezing media. A complete rescue of membrane integrity in freeze-tolerant larvae was observed with a mixture of proline, trehalose, and BSA.

Many cold-acclimated insects accumulate high concentrations of low molecular weight cryoprotectants (CPs) in order to tolerate low subzero temperatures or internal freezing. The sources from which carbon skeletons for CP biosynthesis are driven, and the metabolic reprogramming linked to cold acclimation, are not sufficiently understood. Here we aim to resolve the metabolism of putative CPs by mapping relative changes in concentration of 56 metabolites and expression of 95 relevant genes as larvae of the drosophilid fly, Chymomyza costata transition from a freeze sensitive to a freeze tolerant phenotype during gradual cold acclimation. We found that C. costata larvae may directly assimilate amino acids proline and glutamate from diet to acquire at least half of their large proline stocks (up to 55 µg per average 2 mg larva). Metabolic conversion of internal glutamine reserves that build up in early diapause may explain the second half of proline accumulation, while the metabolic conversion of ornithine and the degradation of larval collagens and other proteins might be two additional minor sources. Next, we confirm that glycogen reserves represent the major source of glucose units for trehalose synthesis and accumulation (up to 27 µg per larva), while the diet may serve as an additional source. Finally, we suggest that interconversions of phospholipids may release accumulated glycero-phosphocholine (GPC) and -ethanolamine (GPE). Choline is a source of accumulated methylamines: glycine-betaine and sarcosine. The sum of methylamines together with GPE and GPC represents approximately 2 µg per larva. In conclusion, we found that food ingestion may be an important source of carbon skeletons for direct assimilation of, and/or metabolic conversions to, CPs in a diapausing and cold-acclimated insect. So far, the cold-acclimation- linked accumulation of CPs in insects was considered to be sourced mainly from internal macromolecular reserves.

Free and open-source software for image analysis and morphological measurements in scientific research is rising in popularity and capabilities as new methods, plugins and macros are being actively developed. A semi-automated method for measuring rat uterus morphology using free and open-source software (Gimp and FIJI) is demonstrated in this paper. Research was performed on ovariectomized rats as a model of osteoporosis (with sham-operated control group). The animals were treated with alendronate, hop extract or the combination of the two. Whole histological slides were photographed and images were manually pre-processed in Gimp. Color masks from Gimp were loaded in FIJI and polar transformation and measurements were made using a custom macro. This analysis was supplemented by manual assessment of Ki67 proliferation marker expression by a pathologist. Our results suggest that monotherapy or combination therapy with alendronate and hop extract does not cause proliferation of the endometrium in ovariectomized rats and would be safe for use in osteoporosis treatment in this regard. The semi-automated method used in this research is more precise and unbiased than older manual methods. Furthermore, it can be easily adapted for analysis of whole-slide images of almost any round or oval organ.

Background/Objectives: New approaches using plant-based extracts are increasingly investigated for osteoporosis treatment, including hop extract, which contains potent compounds that affect bone quality favorably. The objective of this research was to evaluate the effect of alendronate, standardized hop extract and their combination on bone tissue quality, hematological and biochemical parameters, and markers of bone turnover in a rat model of osteoporosis. Methods: The study was performed on 6 month-old female Wistar rats. Sham operation was done on 10 animals, and bilateral ovariectomy on 60 animals. Ovariectomized animals were divided into 6 groups, one not treated, and the others treated with a low dose of alendronate, high dose of alendronate, hop extract, or a combination of low/high dose of alendronate and hop extract. Quality of bone was assessed using micro-CT imaging. Hematology and serum biochemistry were analyzed using standardized kits, and bone turnover markers using Western blot. Results: Treatment with high-dose alendronate, hop extract, and a combination of low-dose alendronate and hop extract significantly improved bone quality in ovariectomized animals, but high-dose alendronate combined with hop extract had no favorable effect. Hematological and biochemical parameters were mostly unaffected and within normal range. Changes in markers of bone turnover were modest. Conclusions: For the first time, the effect of combined use of alendronate with hop extract was analyzed. The combination of a low-dose alendronate with hop extract produced effects comparable to those observed with high-dose alendronate or hop extract alone. Standardized hop extract could be a valuable addition to current osteoporosis therapy.

Background/Objectives: New approaches using plant-based extracts are increasingly investigated for osteoporosis treatment, including hop extract, which contains potent compounds that favorably affect bone quality. The objective of this research was to evaluate the early effect of alendronate, standardized hop extract and their combination on bone tissue quality, hematological and biochemical parameters, and markers of bone turnover in a rat model of osteoporosis. Methods: The study was performed on 6 month-old female Wistar rats. A sham operation was performed on 10 animals, and bilateral ovariectomy on 60 animals. Ovariectomized animals were divided into six groups, one untreated, and the others treated with a low dose of alendronate, high dose of alendronate, hop extract, or a combination of low/high dose of alendronate and hop extract. Bone quality was assessed using micro-CT imaging. Hematology and serum biochemistry were analyzed using standardized kits, and bone turnover markers were measured using Western blot. Results: Treatment with high-dose alendronate, hop extract, and a combination of low-dose alendronate and hop extract significantly improved bone quality in ovariectomized animals, but high-dose alendronate combined with hop extract had no favorable effect. Hematological and biochemical parameters were mostly unaffected and remained within the normal range. Changes in markers of bone turnover were modest. Conclusions: For the first time, combining alendronate with hop extract was analyzed. Short-term treatment with low-dose alendronate and hop extract produced effects comparable to high-dose alendronate or hop extract alone, without clinically relevant alterations in hematological or biochemical parameters. Standardized hop extract may represent a valuable adjunct in osteoporosis therapy.

Background/Objectives: Postmenopausal osteoporosis is associated with systemic metabolic alterations related to estrogen deficiency. This exploratory study investigated the early metabolic, oxidative, and histomorphological effects of alendronate, a standardized hop extract, and their combination in ovariectomized (OV) rats. Methods: Female Wistar rats (n = 70) were randomly allocated to seven groups: six OV groups or a sham-operated control (n = 10 per group). Following a 30-day postoperative period for model development, animals received daily treatment for 2 weeks with vehicle (placebo), low- or high-dose alendronate (1 and 2 mg/kg, respectively), standardized hop extract (60 mg/kg), or both. Oxidative stress markers, liver and perigonadal adipose tissue histology, and tissue metabolism were assessed. Results: No evidence of adverse hepatic, renal, or systemic effects was observed. Oxidative damage markers remained largely unchanged, although ovariectomy was associated with reduced hepatic catalase activity, which was increased by high-dose alendronate. Treatment-related morphological changes in adipose tissue were observed. Serum triglyceride levels were unaffected, whereas total cholesterol was significantly increased in animals receiving hop extract. Hepatic triglyceride levels were influenced by alendronate treatment, modified by hop extract. MALDI-TOF MS suggested no OV-related alterations in amino acid, lipid, steroid, and redox-related metabolism in liver and adipose tissue. The subsequent treatments, especially in the high-dose alendronate group and hop-extract group, partially modified these metabolic signatures; however, these findings remain provisional pending MS/MS validation. Conclusions: Most outcomes were not significantly altered by OV. Alendronate and hop extract exert distinct short-term effects on selected metabolic and oxidative parameters in this exploratory model. Further studies are needed to investigate translational potential.