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The stromal vascular fraction of adipose tissue has gained popularity as regenerative therapy for tissue repair. Both enzymatic and mechanical intraoperative SVF isolation procedures exist. To date, the quest for the preferred isolation procedure persists, due to the absence of standardised yield measurements and a defined clinical threshold. This systematic review is an update of the systematic review published in 2018, where guidelines were proposed to improve and standardise SVF isolation procedures. An elaborate data search in MEDLINE (PubMed), EMBASE (Ovid) and the Cochrane Central Register of Controlled Trials was conducted from September 2016 to date. A total of 26 full‐text articles met inclusion criteria, evaluating 33 isolation procedures (11 enzymatic and 22 mechanical). In general, enzymatic and mechanical SVF isolation procedures yield comparable outcomes concerning cell yield (2.3–18.0 × 105 resp. 0.03–26.7 × 105 cells/ml), and cell viability (70%–99% resp. 46%–97.5%), while mechanical procedures are less time consuming (8–20 min vs. 50–210 min) and cost‐efficient. However, as most studies used poorly validated outcome measures on SVF characterisation, it still remains unclear which intraoperative SVF isolation method is preferred. Future studies are recommended to implement standardised guidelines to standardise methods and improve comparability between studies. [ABSTRACT FROM AUTHOR]

Background and Aims The California Floristic Province (CA-FP) is the most species-rich region of North America north of Mexico. One of several proposed hypotheses explaining the exceptional diversity of the region is that the CA-FP harbours myriad recently diverged lineages with nascent reproductive barriers. Salvia subgenus Audibertia is a conspicuous element of the CA-FP, with multiple sympatric and compatible species. Methods Using 305 nuclear loci and both organellar genomes, we reconstruct species trees, examine genomic discordance, conduct divergence-time estimation, and analyse contemporaneous patterns of gene flow and mechanical reproductive isolation. Key Results Despite strong genomic discordance, an underlying bifurcating tree is supported. Organellar genomes capture additional introgression events not detected in the nuclear genome. Most interfertility is found within clades, indicating that reproductive barriers arise with increasing genetic divergence. Species are generally not mechanically isolated, suggesting that it is unlikely to be the primary factor leading to reproductive isolation. Conclusions Rapid, recent speciation with some interspecific gene flow in conjunction with the onset of a Mediterranean-like climate is the underlying cause of extant diversity in Salvia subgenus Audibertia. Speciation has largely not been facilitated by gene flow. Its signal in the nuclear genome seems to mostly be erased by backcrossing, but organellar genomes each capture different instances of historical gene flow, probably characteristic of many CA-FP lineages. Mechanical reproductive isolation appears to be only part of a mosaic of factors limiting gene flow. [ABSTRACT FROM AUTHOR]

Nanocellulose, an exceptional natural material acquired from cellulose, has received tremendous attention over the past decades. This is because of its unique physical characteristics, surface chemistry, functional properties, and biocompatibility. Cellulose nanocrystals (CNCs) and cellulose nanofibers (CNFs) are two major types of nanocellulose that have received key interest so far. Nanocellulose is commercially produced from wood-based sources. With the ongoing demand for nanocellulose, agricultural wastes and non-woody plants are getting much consideration as cost-efficient alternatives. Water hyacinth (WH) is an alternative source that has shown great prospects for CNC and CNF fabrication. Nanocellulose from WH is proposed for several interesting applications, such as wet-spun filaments, aerogels, packaging films, battery separators, and water filtration, showing promising results. Studies have shown the nanocellulose achieved from WH has a comparable diameter and crystallinity related to the nanocellulose derived from other agricultural wastes, but lower than nanocellulose extracted from wood. Studies were more inclined towards chemical treatments for CNC extraction and mechanical processes for CNF isolation. This review aims to emphasize identifying the true potential of WH as a nanocellulose resource by critical evaluation of the isolation processes, affecting factors on nanocellulose properties, and the foundation of future research for producing sustainable materials. It was perceived that a lot of possibilities remain unexplored as many advanced techniques are yet not well studied for WH nanocellulose extraction. Further, the advantage of being a low-lignin raw material was not properly captured by mapping the more sustainable procedures, which should be a future highlight of WH research. [ABSTRACT FROM AUTHOR]

Background and aimsUnder a widely accepted model of pollinator-driven speciation, geographic variation in pollinator assemblage drives floral divergence and automatically causes reproductive isolation. Yet it is unclear whether divergent floral adaptation initially confers strong reproductive isolation, or whether that comes at later stages of speciation and requires other forms of reproductive isolation. This study uses a pair of recently diverged, interfertile and parapatric species in the genus Clarkia to determine whether adaptation to hawkmoths, a novel pollinator functional group, would automatically confer floral isolation upon sympatric contact.MethodsFirst, genetically based differences in floral traits between C. breweri and C. concinna that would be maintained upon migration are quantified. Then scenarios of experimental sympatry are constructed in which arrays of flowers are exposed to the novel pollinator, the hawkmoth Hyles lineata, and pollinator preference and heterospecific pollen transfer are assessed. Source populations from across the ranges of each species are used to understand how geographic variation in floral traits within species may affect floral isolation.Key resultsAlthough H. lineata has never been observed visiting C. concinna in the wild, it regularly moves between species in experimental floral arrays. Hawkmoth preference and heterospecific pollen transfer vary both among moths and among geographic source locations of C. concinna. The strength of floral isolation in this system is related to variation in flower size, especially hypanthium tube width, and nectar reward among C. concinna forms.ConclusionsAlthough C. breweri has adopted a novel hawkmoth pollination system, both ethological and mechanical floral isolation by hawkmoths are incomplete and vary according to the specific phenotype of the C. concinna source population. The results suggest that strong floral isolation is not automatically conferred by a pollinator shift and may require additional evolution of deterrent floral traits and habitat isolation that reduces the immediate spatial co-occurrence of young species.

Background: Nanofat grafting (NG) is a simple and cost-effective method of lipoaspirates with inter-syringe passages, to produce stromal vascular fraction (SVF) and isolate adipose-derived stem cells (ASCs). This represents a tremendous interest in the future clinical needs of tissue engineering. In this study, we optimized the NG technique to increase the yield of ASC extractions.Methods: We analyzed three groups of SVF obtained by 20, 30, and 40 inter-syringe passages. The control group was an SVF obtained by enzymatic digestion with Celase. We studied their cell composition by flow cytometry, observed their architecture by confocal microscopy, and observed immunomodulatory properties of the ASCs from each of the SVFs by measuring inflammatory markers of macrophages obtained by an ASC monocyte co-culture.Results: We have established the first cell mapping of the stromal vascular fraction of adipose tissue. The results showed that SVF obtained by 20 inter-syringe passages contains more statistically significant total cells, more cells expressing the ASC phenotype, more endothelial cells, and produces more CFU-F than the SVF obtained by 30 and 40 passages and by enzymatic digestion. Confocal microscopy showed the presence of residual adipocytes in SVF obtained by inter-syringe passages but not by enzymatic digestion. The functional study indicates an orientation toward a more anti-inflammatory profile and homogenization of their immunomodulatory properties.Conclusion: This study places mechanically dissociated SVF in the center of approaches to easily extract ASCs and a wide variety and number of other progenitor cells, immediately available in a clinical setting to provide both the amount and quality of cells required for decellularized tissues.

At present, the primary culture method of microglia is complicated, and the culture of spinal cord microglia is rare, so we will explore to establish a new and efficient primary culture method of microglia in rats with spinal cord injury (SCI). The SCI model of SD rats was established by modified A11en′s method, and the model of SCI was performed on 1 d, 3 d, 7 d and 14 d respectively. Then the injured spinal cord was removed, mechanically separated and filtered. The morphology of microglia was observed the next day and its purity was identified by CD11b and Iba1 immunofluorescence labeling. According to the above results, the morphological changes of microglia after 3 d of SCI were observed at 1 d, 2 d and 4 d. The results showed that the purity of microglia was 98%. The number of microglia after 3 d of SCI was the most. After SCI, the migration ability of microglia was enhanced, the number of microglia in the injured area increased, and the number was the highest at 3 d, then gradually decreased. In addition, the microglia after SCI would gradually change from active state to resting state with the passage of time. Therefore, we can use a simple and efficient mechanical separation method to extract primary microglia, which provides the basis for the study of microglia. 1. The method solves the difficulty of extracting microglia for a long time. 2. The method provides the possibility for dynamic detection of microglia. 3. The method can obtain high purity microglia efficiently and economically. 4. We can use a simple mechanical separation method to extract primary microglia. [ABSTRACT FROM AUTHOR]

An increased divergence in characters between species in secondary contact can be shaped by selection against competition for a common resource (ecological character displacement, ECD) or against maladapted hybridization (reproductive character displacement, RCD). These selective pressures can act between incipient species (reinforcement) or well-separated species that already completed the speciation process, but that can still hybridize and produce maladapted hybrids. Here, we investigated two well-separated sexually deceptive orchid species that, unusually, share their specific pollinator. Sympatric individuals of these species are more divergent than allopatric ones in floral characters involved in a mechanical isolating barrier, a pattern suggestive of RCD. To experimentally test this scenario, we built an artificial sympatric population with allopatric individuals. We measured flower characters, genotyped the offspring in natural and artificial sympatry and estimated fertility of hybrids. Different from naturally sympatric individuals, allopatric individuals in artificial sympatry hybridized widely. Hybrids showed lower pollination success and seed viability than parentals. Character displacement did not affect plant pollination success. These findings suggest that RCD evolved between these species to avoid hybridization and that selection on reinforcement may be very strong even in plants with highly specialized pollination. [ABSTRACT FROM AUTHOR]

Mechanically processed stromal vascular fraction (mSVF) is a promising source for regenerative purposes. To study the in vivo fate of the mSVF, we herein used a vascularized tissue engineering chamber that insulates the target mSVF from the surrounding environment. In contrast to previous models, we propose an arteriovenous (AV) shunt between saphenous vessels in rats without a venous graft. Mechanical SVF was processed from the fat pads of male Sprague Dawley rats, mixed with a fibrin hydrogel and implanted into an inguinal tissue engineering chamber. An arteriovenous shunt was established between saphenous artery and vein. On the contralateral side, an mSVF-fibrin hydrogel mix without vascular axis served as a non-vascularized control. After two and six weeks, rats were sacrificed for further analysis. Mechanical SVF showed significant numbers of mesenchymal stromal cells. Vascularized mSVF explants gained weight over time. Perilipin and CD31 expression were significantly higher in the mSVF explants after six weeks while no difference in DAPI positive cells, collagen deposition and FABP4 expression was observed. Morphologically, no differentiated adipocytes but a dense cell-rich tissue with perilipin-positive cells was found after six weeks. The phosphorylation of ERK1/2 was significantly enhanced after six weeks while Akt activation remained unaltered. Finally, mSVF explants stably expressed and released VEGF, bFGF and TGFb. Vascularized mSVF is able to proliferate and express adipocyte-specific markers. The AV shunt model is a valuable refinement of currently existing AV loop models in the rat which contributes to the fundamental 3R principles of animal research.

Stomata, which are microscopic valves on the leaf surface formed by two guard cells (GC), play a critical role in the regulation of leaf water and gas exchange and, hence, determine plant adaptive potential. However, little data is available on GC biochemistry, protein abundance and gene expression, mainly due to technical difficulties and challenges in isolating sufficient amounts of high-quality pure GC. In the present study we applied some modifications to the mechanical isolation of guard-cell to generalise this method for diverse growth conditions as well as plant species. Epidermal peel fragments enriched in guard cells were mechanically isolated from quinoa, spinach and sugar beet leaves grown at two conditions (normal and salt stress). Multiple analysis was performed to confirm the suitability and superiority of the modified technique to the original method. At the first step, the viability and purity of GC-enriched epidermal fragments were assessed under the microscope. Then, the RNA integrity, gene expression, and 1D SDS-PAGE tests were performed to validate the suitability of this technique for omics studies. The data revealed a wide range of proteins as well as a high integrity of RNA extracted from guard cell samples. The expression level of several GC-specific genes and mesophyll-dominant genes were investigated using a comparative analysis of transcriptome datasets of GC and whole-leaf samples. We found that Rubisco and photosynthesis-related proteins such as chlorophyll a / b binding protein were substantially higher in the whole leaf compared with the GCs. More importantly, GC-specific genes such as OST1 , SLAC1 , MYB60 , FAMA and HT1 were highly expressed in the GCs, confirming that our guard cell preparation was highly enriched in GC gene transcripts. Real-time quantitative reverse transcription PCR further confirmed the efficacy of the GC isolation technique for exploring responses of GC to diverse types of stress at the molecular level. Guard cells (GCs) play a critical role in the regulation of leaf water and gas exchange in plants. Due to technical difficulties in GC isolation, little data is available on molecular studies of GC. Here, we develop an optimised method from the leaves of spinach, sugar beet and quinoa under control and salt-stress conditions and provide direct evidence to test the quality of samples. [ABSTRACT FROM AUTHOR]

Lignin-containing cellulose nanofibers (LCNFs) with promising sustainability and multiple functionalities are highly in demand while still limited by the high energy consumption and potential environment disruption in preparation. In this work, the super-base system (dimethyl sulfoxide/potassium hydroxide, DMSO/KOH) was firstly employed as pseudosolvent to swell melon vine powder by partially remove some non-cellulose components (such as extraction, lignin and ash) in plant cell wall, and facilitate the following production of LCNFs. Due to the strong nucleophilicity of DMSO/KOH, universal swollen effects toward corn husk, poplar, bagasse, rice husk, and cotton stalk were achieved, and the LCNFs from them were also successfully obtained (with yields of ∼78.75%, 55.60%, 51.33%, 46.13%, and 53.90%, respectively). Besides, this method resulted in high lignin content among LCNFs (∼19.54%), which endowed the resultant membranes with superior mechanical strength, thermal stability, and dye rejection performance (a rejection rate of ∼92.7%, and a flux of 15.16 L•m-2•h-1•bar-1 toward methyl violet at 250 ppm). Thus, this research paves a simple, efficient, and universal way to prepare LCNFs from various biomass. [Display omitted] • DMSO/KOH system was firstly employed to facilitate the production of LCNFs. • DMSO/KOH was suitable for LCNFs production from various biomasses. • The LCNFs membranes exhibited outstanding dye rejection performances. [ABSTRACT FROM AUTHOR]

Over the last decades, there has been ongoing and evolving research concerning regenerative medicine, specifically, stem cells. The most common source of adult mesenchymal stem cells (MSCs) remains the adipose tissue and the easiest way to obtain such tissue is lipoaspirate. The fatty tissue obtained can be processed either in an enzymatic way, which is time-consuming and expensive and carries several dangers for the viability of the stem cells included, or with mechanical means which are fast, inexpensive, yield enough viable cells, and can be readily used for autologous transplantation in one-stage procedures. Herein, we demonstrate our non-enzymatic method for obtaining adipose-derived stromal vascular fraction comprising MSCs. The stromal vascular fraction was isolated via centrifugation, and the characteristics and numbers of the cells isolated have been tested with flow cytometry assay, cell culture, and differentiation. Over 91% of viable MSCs were isolated using the mechanical method. The cells retained the ability to differentiate into osteocytes, adipocytes, and chondrocytes. The method presented is simple, requiring no special equipment, and yields a viable population of stem cells in large numbers. These cells can be readily used in several operations (orthopedic, dentistry, fistulas, etc.) making feasible "one-stage" procedures, thus proving their benefits for the patient and the health care system., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2024, Goulas et al.)

This article is a Commentary on Minnaar et al., 224: 1160–1170. [ABSTRACT FROM AUTHOR]

Summary: The causative link between phenotypic divergence and reproductive isolation is an important but poorly understood part of ecological speciation. We studied the effects of floral‐tube length variation on pollen placement/receipt positions and reproductive isolation.In a population of Lapeirousia anceps (Iridaceae) with bimodal floral‐tube lengths, we labelled pollen of short‐ and long‐tubed flowers with different colour fluorescent nanoparticles (quantum dots). This enabled us to map pollen placement by long‐ and short‐tubed flowers on the only floral visitor, a long‐proboscid fly. Furthermore, it allowed us to quantify pollen movement within and between short‐ and long‐tubed flowers.Short‐ and long‐tubed flowers placed pollen on different parts of the pollinator, and long‐tubed flowers placed more pollen per visit than short‐tubed flowers. This resulted in assortative pollen receipt (most pollen received comes from the same phenotype) and strong but asymmetric reproductive isolation, where short‐tubed plants are more reproductively isolated than long‐tubed plants.These results suggest that floral‐tube length divergence can promote mechanical isolation in plants through divergence in pollen placement sites on pollinators. Consequently, in concert with other reproductive isolation mechanisms, selection for differences in floral‐tube length can play an important role in ecological speciation of plants. See also the Commentary on this article by Machhala, 224: 1005–1008. [ABSTRACT FROM AUTHOR]

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The mammalian ovary is a substantial source of oocytes arranged into follicles at various stages of folliculogenesis, from the primordial to the ovulatory ones. Primordial follicles constitute the most abundant source of gametes inside the mammalian ovary at any given time.The isolation of a high number of primordial follicles, together with the development of protocols for in vitro follicle growth, would provide a powerful tool to fully exploit the female reproductive potential and boost the rescue and restoration of fertility in assisted reproduction technologies in human medicine, animal breeding, and preservation of threatened species. However, the most significant limitation is the lack of efficient methods for isolating a healthy and homogeneous population of viable primordial follicles suitable for in vitro culture. Here, we provide a fast and high-yield strategy for the mechanical isolation of primordial follicles from limited portions of the ovarian cortex in the bovine animal model., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

This paper presents the design of stretchable elastomer-liquid metal capacitors fabricated with mechanical isolation structures. We envision potential application to stretchable and strain stable devices such as chemical sensors, as well as temperature compensation in soft sensors. A strength of the employed isolation approach is the ability to fabricate strain sensitive and strain isolated capacitors in the same substrate. A finite element model is presented, and the responses to $x$ -axis strain, $y$ -axis strain, and temperature are characterized. The capacitor with a labyrinth isolation structure shows a five-fold reduction in $x$ -axis strain response over a capacitor without isolation. Co-fabricated strain sensitive and strain isolated capacitors show a factor-of-four reduction in temperature response over the strain sensitive capacitor, demonstrating potential applications as a temperature compensated and stretchable strain or pressure sensors. [ABSTRACT FROM AUTHOR]

Isolation barriers work at different instances during the mating process in odonate insects. In territorial damselflies, heterospecific interactions are mainly precluded by sexual (visual) isolation, while in non-territorial damselflies, heterospecific interactions are mostly precluded by mechanical isolation and sexual (tactile) isolation. In this study we investigated the strength of three premating barriers (visual, mechanical and tactile), genetic divergence and degree of sympatry (on their entire distribution) between four non-territorial Argia damselflies (A. anceps, A. extranea, A. oenea and A. tezpi). Our results are explained in the light of learned mating preferences and Kaneshiro's hypothesis. We detected a strong reproductive isolation between all pairs of species by the joint action of the three studied barriers [visual (90.6%), mechanical (8.7%) and tactile (0.7%)]. Sexual (visual) isolation was the most important barrier, perhaps driven by learning mating preferences. One of the studied species, A. extranea, which is the most derived of the studied species, showed a highly asymmetric isolation in reciprocal crosses, which is consistent with Kaneshiro's hypothesis. Moreover, we detected a negligible ecological niche differentiation between the studied species (70% of shared distribution). Our results suggest that sexual (visual) selection may be an important force driving speciation in non-territorial species. [ABSTRACT FROM AUTHOR]

Industrial hemp is a green economic plant for biogas and fuel. It is also used mainly in construction, paper, and textile industries. After harvested, the stubs of hemp are left as wastes. Therefore, the added value of this part is considered in this study. Mechanical defibrillation is an interesting method which does not require any harmful chemicals to be involved for size reduction. In this work, high-pressure homogenization by microfluidizer was carried out to defibrillate hemp fibers. Three investigating parameters, which were chemical pretreatment (delignification and hemicellulose removal), fiber concentration, and defibrillation time, were studied. It was found that chemical pretreatment enhanced capability of mechanical defibrillation in which nanofibrillated cellulose could be obtained shortly by microfluidizer (starting from only 15 min defibrillation to obtain 20–50 nm fiber diameters). Higher fiber concentration during defibrillation provided greater degree of polymerization and slightly higher thermal degradation temperature. Meanwhile, longer defibrillation time resulted in smaller and more uniformity of size of nanofibers and provided significant lower degree of polymerization and minor decrease in thermal degradation temperature. However, no significant change on crystallinity was observed. From the obtained results, 0.7% w/v of fiber concentration and 120 min defibrillation time using chemi-mechanical method provided the most uniformity of fiber size and the highest transparency of an aqueous suspension as well as its cast film. [ABSTRACT FROM AUTHOR]

Systematists and geneticists study biological diversity, but they use different approaches that rarely intersect. A very common pattern that is of interest for both researchers is the rapid evolution of genitalia, a trait of significant taxonomic utility in several sexually reproducing animal clades. The idea that both male and female genitalia are species-specific and play a role in reproductive isolation has long been controversial but has recently gained a renewed interest by speciation and developmental geneticists. Here, I highlight six unresolved questions in genitalia coevolution and I argue that systematists, with their well training in comparative morphology, usage of large and geographically diverse collections, and ability to apply molecular genetics techniques, can make important contributions. Such an extension of systematics into the speciation and developmental genetics realms is a promising opportunity to expand 'integrative taxonomy' comparisons between DNA and morphology into more explanatory relationships between the two sources of taxonomic data. [ABSTRACT FROM AUTHOR]

The interaction between floral traits and reproductive isolation is crucial to explaining the extraordinary diversity of angiosperms. Heterostyly, a complex floral polymorphism that optimizes outcrossing, evolved repeatedly and has been shown to accelerate diversification in primroses, yet its potential influence on isolating mechanisms remains unexplored. Furthermore, the relative contribution of pre- versus postmating barriers to reproductive isolation is still debated. No experimental study has yet evaluated the possible effects of heterostyly on pre- and postmating reproductive mechanisms. We quantify multiple reproductive barriers between the heterostylous Primula elatior (oxlip) and P. vulgaris (primrose), which readily hybridize when co-occurring, and test whether traits of heterostyly contribute to reproductive barriers in unique ways. We find that premating isolation is key for both species, while postmating isolation is considerable only for P. vulgaris; ecogeographic isolation is crucial for both species, while phenological, seed developmental, and hybrid sterility barriers are also important in P. vulgaris, implicating sympatrically higher gene flow into P. elatior. We document for the first time that, in addition to the aforementioned species-dependent asymmetries, morph-dependent asymmetries affect reproductive barriers between heterostylous species. Indeed, the interspecific decrease of reciprocity between high sexual organs of complementary floral morphs limits interspecific pollen transfer from anthers of short-styled flowers to stigmas of long-styled flowers, while higher reciprocity between low sexual organs favors introgression over isolation from anthers of long-styled flowers to stigmas of short-styled flowers. Finally, intramorph incompatibility persists across species boundaries, but is weakened in long-styled flowers of P. elatior, opening a possible backdoor to gene flow through intramorph pollen transfer between species. Therefore, patterns of gene flow across species boundaries are likely affected by floral morph composition of adjacent populations. To summarize, our study highlights the general importance of premating isolation and newly illustrates that both morph- and species-dependent asymmetries shape boundaries between heterostylous species. [ABSTRACT FROM AUTHOR]

Adaptive radiations have long served as living libraries to study the build-up of species richness; however, they do not provide good models for radiations that exhibit negligible adaptive disparity. Here, we review work on damselflies to argue that nonadaptive mechanisms were predominant in the radiation of this group and have driven species divergence through sexual selection arising from male-female mating interactions. Three damselfly genera (Calopteryx, Enallagma and Ischnura) are highlighted and the extent of (i) adaptive ecological divergence in niche use and (ii) nonadaptive differentiation in characters associated with reproduction (e.g. sexual morphology and behaviours) was evaluated. We demonstrate that species diversification in the genus Calopteryx is caused by nonadaptive divergence in coloration and behaviour affecting premating isolation, and structural differentiation in reproductive morphology affecting postmating isolation. Similarly, the vast majority of diversification events in the sister genera Enallagma and Ischnura are entirely driven by differentiation in genital structures used in species recognition. The finding that closely related species can show negligible ecological differences yet are completely reproductively isolated suggests that the evolution of reproductive isolation can be uncoupled from niche-based divergent natural selection, challenging traditional niche models of species coexistence. [ABSTRACT FROM AUTHOR]

Stigmaeopsis celarius Banks (hereafter Sc) is a spider mite living and feeding on the leaves of various bamboo species such as Moso bamboo [ Phyllostachys edulis (= P. pubescens)] and Pleioblastus spp. (Poaceae). A previous phylogenetic study revealed a cryptic, phylogenetic sister species to Sc (hereafter Ss). Although its life type appears to be similar to that of Sc, individuals of Ss make much smaller nests compared with Sc, and the nests have been found mostly on Nezasa bamboo ( Pleioblastus argenteostriatus). To investigate whether Sc and Ss are reproductively isolated, we explored their populations in southwestern Japan, and crossed them to examine mating behaviors and fertilization success. Field surveys revealed that the nests of these two species occur on the same leaves and, thus, the individuals of these species may make frequent contact. Reciprocal crosses suggested that the two species are reproductively isolated. Though Sc males have tried to mate with Ss females, copulation seldom occurred because of their long opisthosoma (hind body), which prevented the insertion of the aedeagus into the genitalia of Ss females. In contrast, most Ss males ignored Sc females, and eggs were not fertilized even in the few cases where copulation appeared to occur. These results suggest that strong selection pressure is imposed on body length to prevent interspecific hybridization in the contact area of these species. [ABSTRACT FROM AUTHOR]

Background and Aims Plants surrounded by individuals of other co-flowering species may suffer a reproductive cost from interspecific pollen transfer (IPT). However, differences in floral architecture may reduce or eliminate IPT. Methods A study was made of Pedicularis densispica (lousewort) and its common co-flowering species, Astragalus pastorius, to compare reproductive and pollination success of lousewort plants from pure and mixed patches. Floral architecture and pollinator behaviour on flowers of the two plants were compared along with the composition of stigmatic pollen load of the louseworts. The extent of pollen limitation of plants from pure and mixed patches was also explored through supplemental pollination with self- and outcross pollen (PLs and PLx). Key Results Mixed patches attracted many more nectar-searching individuals of Bombus richardsi. These bumble-bees moved frequently between flowers of the two species. However, they pollinated P. densispica with their dorsum and A. pastorius with their abdomen. This difference in handling almost completely eliminated IPT. Lousewort plants from mixed patches yielded more seeds, and seeds of higher mass and germinability, than those from pure patches. Moreover, louseworts from mixed patches had lower PLs and PLx compared with those from pure patches. Conclusions Differences in floral architecture induced differences in pollinator behaviour that minimized IPT, such that co-flowering plants significantly enhanced quantity and quality of pollinator visits for the lousewort plants in patchy habitat. These findings add to our understanding of the mechanisms of pollination facilitation. [ABSTRACT FROM AUTHOR]

Follicles are the functional unit of the ovary and several methods have been developed to grow follicles ex vivo, which recapitulate key events of oogenesis and folliculogenesis. Enzymatic digestion protocols are often used to increase the yield of follicles from the ovary. However, the impact of these protocols on the outermost theca and granulosa cells, and thereby follicle function, is not well defined. To investigate the impact of enzymatic digestion on follicle function, we collected preantral follicles from CD1 mice either by enzymatic digestion (Enzy-FL) or mechanical isolation (Mech-FL) and compared follicle growth, steroidogenesis and cell differentiation within an encapsulated in vitro follicle growth system which maintains the 3D architecture of the oocyte and its surrounding somatic cells. Follicles were encapsulated in 0.5% alginate and cultured for 8 days. Compared with Enzy-FL, Mech-FL grew more rapidly and produced significantly higher levels of androstenedione, estradiol and progesterone. The expression of theca-interstitial cell marker genes, Cyp17a1, which encodes 17-hydroxylase/17, 20-lyase and catalyzes the hydroxylation of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone, and the conversion of these products into dehydroepiandrosterone and androstenedione, and Star, which encodes a transport protein essential for cholesterol entry into mitochondria, were also higher in Mech-FL than in Enzy-FL. Mech-FL maintained an intact theca-interstitial layer on the outer edge of the follicle that phenocopied in vivo patterns as confirmed by alkaline phosphatase staining, whereas theca-interstitial cells were absent from Enzy-FL from the onset of culture. Therefore, preservation of the theca cell layer at the onset of culture better supports follicle growth and function. Interestingly, granulosa cells in the outermost layers of Enzy-FL expressed CYP17A1 by Day 4 of culture while maintaining inhibin α-subunit expression and a cuboidal nucleus. Thus, in the absence of theca-interstitial cells, granulosa cells have the potential to differentiate into androgen-producing cells. This work may have implications for human follicle culture, where enzymatic isolation is required owing to the density of the ovarian cortex., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Gene flow between hybridizing plant species depends strongly on pollinator behaviour, which affects pollen transfer among floral types and reproductive isolation. We examined bumblebee behaviour and pollen transfer between two hybridizing Rhinanthus species that are very similar in ecology and floral traits. The two species, Rhinanthus minor and R. angustifolius, shared similar pollinator guilds and assemblages, but pollinator recruitment and flower visitation rates were higher in R. angustifolius sites, probably because of its higher reward levels and better visibility. When presented with Rhinanthus flowers, bumblebees that previously foraged on R. angustifolius were less prone to visit R. minor inflorescences, while R. minor foragers accepted both species in similar proportions. Although Rhinanthus has been cited as a case of mechanical isolation resulting from interactions between bee behaviour and differences in stigma and anther placement, we found no support for efficient mechanical reproductive isolation. Bumblebees that foraged on R. minor flowers carried more pollen, but pollen placement on their bodies was similar to that of bees that visited R. angustifolius, and cross-specific stigmatic pollen deposition was similar in both directions. However, the asymmetry in pollinator handling time between the two species, due to dissimilar pollen rewards, may have lowered relative heterospecific pollen receipt on R. angustifolius, suggesting that net gene flow resulting from pollen transfer dynamics is more likely towards R. minor, although this effect remains weak and will be most likely counterbalanced by context-based labile pollinator preference. [ABSTRACT FROM AUTHOR]

Mechanical reproductive barriers have been dismissed as a major driver of animal speciation, yet the extent to which such barriers cause reproductive isolation in most animal groups is largely unknown and rarely tested. In this study, we used hierarchical Bayesian modeling of mate compatibility experiments to show that body size divergence in lizards of the Plestiodon skiltonianus complex contributes to reproductive isolation in at least three ways: males preferably court females that are more similar in size, females reject males that are highly divergent in size, and the size difference of a male and female in copula constrains the ability to align the genitalia for intromission. We used a predictive model to estimate the contributions of behavioral and mechanical barriers to reproductive isolation between populations with differing degrees of size divergence. This model shows that the mechanical barrier is more important than behavioral barriers at small and intermediate degrees of size divergence, suggesting that it acts earlier during speciation when body morphology is more similar between diverging lineages. Given that correlated divergence in size and ecology is common in animals, similar constraints imposed by the geometry of the mating posture may apply to a variety of major animal lineages and merit further attention in speciation research. [ABSTRACT FROM AUTHOR]

Background and Aims Increasing evidence challenges the conventional perception that orchids are the most distinct example of floral diversification due to floral or prezygotic isolation. Regarding the relationship between co-flowering plants, rewarding and non-rewarding orchids in particular, few studies have investigated whether non-rewarding plants affect the pollination success of rewarding plants. Here, floral isolation and mutual effects between the rewarding orchid Galearis diantha and the non-rewarding orchid Ponerorchis chusua were investigated. Methods Flowering phenological traits were monitored by noting the opening and wilting dates of the chosen individual plants. The pollinator pool and pollinator behaviour were assessed from field observations. Key morphological traits of the flowers and pollinators were measured directly in the field. Pollinator limitation and interspecific compatibility were evaluated by hand-pollination experiments. Fruit set was surveyed in monospecific and heterospecific plots. Key Results The species had overlapping peak flowering periods. Pollinators of both species displayed a certain degree of constancy in visiting each species, but they also visited other flowers before landing on the focal orchids. A substantial difference in spur size between the species resulted in the deposition of pollen on different regions of the body of the shared pollinator. Hand-pollination experiments revealed that fruit set was strongly pollinator-limited in both species. No significant difference in fruit set was found between monospecific plots and heterospecific plots. Conclusions A combination of mechanical isolation and incomplete ethological isolation eliminates the possibility of pollen transfer between the species. These results do not support either the facilitation or competition hypothesis regarding the effect of nearby rewarding flowers on non-rewarding plants. The absence of a significant effect of non-rewarding P. chusua on rewarding G. diantha can be ascribed to low levels of overlap between the pollinator pools of two species. [ABSTRACT FROM AUTHOR]

Objective To compare the effects of mechanical isolation and pancreatin digestant isolation in separating neural stem cells (NSCs) from brain tissues of neonatal rats. Methods The cerebral cortex of newborn rals with in 3 days were isolated and cultured by mechanical and panerealin digestion, respectively. The NSCs were identified and differentiated by immunofluorescence assay. Diameter and number of cell spheres were detected on day 2, 5 and 7 of culture. The proliferation ability of the primary passage of NSCs and ihe third-passage NSCs in the two groups were compared by MTT. The third-passage NSCs were frozen and recovered and the survival rate of the cells was calculated. Results Both of these two methods could isolate NSCs from the cerebral cortex of neonatal rats, and those NSCs could be induced to differentiate into neurons and glial cells. On day 5 and 7, the NSCs' ball number and diameter which came from the mechanical isolation group were higher than those of the pancreatin digestant group in the same culture time (all P < 0.05). On day 2, 5 and 7, the prime NSCs which came from ihe mechanical isolation group had better clonal proliferation ability than those of the pancreatin digestant group in the same culture time (all P < 0.05). On day 2 and 5, ihe ihird-passage NSCs of the mechanical isolation group had better clonal proliferation ability than those of the pancreatin digestant group in the same culture time (all P <0.05). The survival rates of the third-passage NSCs of the mechanical isolation group and pancreatin digestanl group were respectively 85% and 89%, respectively ; and there was no significant difference between these two groups (P>0. 05). Conclusion Both mechanical isolation and pancreatin digestant isolation can successfully isolate NSCs. NSCs which come from the mechanical isolation group have better clonal proliferation ability than those of the pancreatin digestant group. [ABSTRACT FROM AUTHOR]

Abstract: The present study aimed to analyze different methods of mechanical isolation of buffalo preantral follicles (Experiment I), in vitro culture of isolated follicles in different groups of culture medium over collagen gel matrix (Experiment II) and subsequent in vitro development and survival of isolated preantral follicles (PFs) (Experiment III). In Experiment I, ovarian cortical pieces were separated and PFs isolated by different mechanical methods. In Experiment II, isolated follicles were divided into three groups and cultured in TCM-199 having 10% FBS, 1% ITS, and 20 ng/ml EGF (Group A, control), addition of 0.5 μg/ml FSH (Group B) or FSH+100 ng/ml IGF-I (Group C). Follicles were incubated at 38.5 °C in 5% CO2 with maximum humidity. In Experiment III, based on the outcome of Experiment I and II, PFs were cultured from those isolation method and treatment group, showing better growth and developmental pattern to analyze the impact of growth factors on in vitro growth of follicles in long term culture. It was found that micro-dissected PFs showed higher survival rate and growth after 15 days of culture compared to PFs isolated by other methods. Follicles cultured with FSH+IGF-1 on collagen gel matrix, showed significantly (P <0.05) higher survival rate and mean diameter of follicles on day 15 of culture compared to control. In summary, it has been shown that isolation of follicles by micro-dissection has advantages over other methods, being relatively simple, inexpensive and less harmful to follicles. Micro-dissected buffalo PFs maintained the architecture, showed antrum formation in presence of FSH and IGF-I over the collagen gel matrix. [Copyright &y& Elsevier]

The role of species-specific genitalia in reproductive isolation is unclear. Males of the millipede genus Parafontaria use gonopods (modified eighth legs) charged with sperm from the genital openings of the second legs as intromittent organs. Males perform both preliminary and true intromission during mating. During preliminary intromission, a male attempts to insert his gonopods into the female genitalia before charging the gonopods with sperm. If this intromission is completed, it is followed by the ejaculation of sperm to the gonopods and true intromission for insemination. In two sympatric species of Parafontaria that lack effective precopulatory isolation, copulation was terminated without insemination because of preliminary intromission failure caused by mismatched genital and body sizes. Thus, mechanical isolation between these sympatric species resulted from morphological differentiation mediated by the obligatory preliminary intromission. These findings demonstrate the proximate importance of genital and body size differences for reproductive isolation within this genus of millipede. [ABSTRACT FROM AUTHOR]

Abstract: A method for selectively isolating and aggregating intact nanotubes from leaf surfaces, in sample quantities useable for their characterisation in further experimental investigations, is presented here. It uses liquid sucrose, as a saturated solution, with its wetting behaviour modified by the addition of controlled amounts of ethanol, as a temporary entrapment and release medium, for removing nanotube material from the leaf surface, here of Picea pungens (Engelmann). This harvesting technique works by the slow, gentle transition of the medium from liquid to solid, capturing the nanotubes, and then back to liquid again, releasing them, to form an aggregate sample, with little compromise to the structural integrity of individual nanotubes. Scanning electron microscopy (SEM) images are presented rigorously illustrating the technique and its effectiveness. Comparison with other recent methods reveals its advantages, and potential applications are explored. [Copyright &y& Elsevier]

The shapes and lengths of copulatory pieces and vaginal appendices of the carabid beetle subgenus Ohomopterus (genus Carabus) vary among species. In Japan, the species in the group with a medium body size ( C. yaconinus, C. iwawakianus, C. maiyasanus, C. uenoi, C. arrowianus, C. esakii, and C. insulicola) are usually allopatric or parapatric, except at Mt Kongosan, where C. uenoi, C. iwawakianus, and C. yaconinus are sympatrically distributed. The degree of premating isolation by mate preference was high between sympatric populations, irrespective of the genetic distance between them. However, premating isolation was absent between parapatric populations. The degree of premating isolation for allopatric populations spanned a wide range of isolation values. Thus, mate discrimination by males seems to have evolved mostly between sympatric pairs. These results suggest two hypotheses. First, premating isolation has evolved through reinforcement or through reproductive character displacement after sympatric contact. Second, premating isolation has evolved in allopatry, and as a result of premating isolation, the species can coexist in sympatry. We also examined the degree of mechanical isolation between C. uenoi and C. iwawakianus (a sympatric pair), which have a very large difference in the length of the copulatory piece. The insertion success was low and only one female produced viable offspring among 15 crosses; however, death in females due to copulation was rare. For sympatric matings between C. uenoi and C. iwawakianus, a large difference in the genital size might reduce the gene flow with small mating costs. Gene flow that was significantly reduced by genital difference might cause either the evolution of premating isolation through reinforcement/reproductive character displacement or through the maintenance of a high degree of premating isolation following sympatric contact. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 87, 145–154. [ABSTRACT FROM AUTHOR]

Adipose-derived Stem Cells (ASCs) present great potential for reconstructive procedures. Currently, isolation by enzyme digestion and culturing using xenogenic substances remain the gold standard, impairing clinical use. Abdominal lipo-aspirate and blood samples were obtained from healthy patients. A novel mechanical isolation method for ASCs was compared to (the standard) collagenase digestion. ASCs are examined by flowcytometry and multilineage differentiation assays. Cell cultures were performed without xenogenic or toxic substances, using autologous plasma extracted from peripheral blood. After eGFP-transfection, an in vivo differentiation assay was performed. Mechanical isolation is more successful in isolating CD34+/CD31−/CD45−/CD13+/CD73+/CD146− ASCs from lipo-aspirate than isolation via collagenase digestion (p <0.05). ASCs display multilineage differentiation potential in vitro. Autologous plasma is a valid additive for ASCs culturing. eGFP-ASCs, retrieved after 3 months in vivo, differentiated in adipocytes and endothelial cells. A practical method for human ASC isolation and culturing from abdominal lipo-aspirate, without the addition of xenogenic substances, is described. The mechanical protocol is more successful than the current gold standard protocol of enzyme digestion. These results are important in the translation of laboratory-based cell cultures to clinical reconstructive and aesthetic applications. • Non-enzymatic isolation method for ASCs is compared to standard collagenase digestion. • Mechanical isolation of ASCs is more successful than via collagenase digestion. • Isolated ASCs display multilineage differentiation potential in vitro. • Autologous plasma is a valid additive for ASCs culturing. [ABSTRACT FROM AUTHOR]

Adaptive radiations have long served as living libraries to study the build-up of species richness; however, they do not provide good models for radiations that exhibit negligible adaptive disparity. Here, we review work on damselflies to argue that nonadaptive mechanisms were predominant in the radiation of this group and have driven species divergence through sexual selection arising from male-female mating interactions. Three damselfly genera (Calopteryx,Enallagma and Ischnura) are highlighted and the extent of (i) adaptive ecological divergence in niche use and (ii) nonadaptive differentiation in characters associated with reproduction (e.g. sexual morphology and behaviours) was evaluated. We demonstrate that species diversification in the genus Calopteryx is caused by nonadaptive divergence in coloration and behaviour affecting premating isolation, and structural differentiation in reproductive morphology affecting postmating isolation. Similarly, the vast majority of diversification events in the sister genera Enallagma and Ischnura are entirely driven by differentiation in genital structures used in species recognition. The finding that closely related species can show negligible ecological differences yet are completely reproductively isolated suggests that the evolution of reproductive isolation can be uncoupled from niche-based divergent natural selection, challenging traditional niche models of species coexistence.

This study was designed to investigate an effective method for the derivation of human embryonic stem cells (hESC) from poor quality embryos discarded from in vitro fertilization (IVF) laboratories. Poor quality embryos were donated from IVF center on day 3 and cultured in a blastocyst medium for 2 days, and then in a blastocyst optimum culture medium for additional 2 days. The isolated inner cell masses (ICM) were inoculated onto the feeder layer of mouse embryonic fibroblasts for sub cultivation. The biological characterizations and differentiation capability of these cells were examined after at least 15 passages. Forty-two embryos developed to expanded blastocysts or hatched blastocysts and ICMs were successfully isolated from all embryos. Six new hESC lines were established after further culture. All hESC lines were positive for stage-specific embryonic antigen SSEA-4, TRA-1-81, TRA-1-60 markers and negative for SSEA-1 marker. They showed a high level of alkaline phosphatase activity, positive expression of transcription factor Oct-4. Furthermore, these cells had the potential to form both embryoid bodies in vitro and teratomas in vivo. Five hESC lines had normal karyotypes and one had trisomy 13. In this paper we described a stable and effective system for hESC isolation and established six new hESC lines, which will provide an excellent experimental model for cell therapy, developmental biology and genetic research. [ABSTRACT FROM AUTHOR]