Crowdfunding for health research: a qualitative evidence synthesis and a pilot programme.

BACKGROUND: Many low-income and middle-income country (LMIC) researchers have disadvantages when applying for research grants. Crowdfunding may help LMIC researchers to fund their research. Crowdfunding organises large groups of people to make small contributions to support a research study. This manuscript synthesises global qualitative evidence and describes a Special Programme for Research and Training in Tropical Diseases (TDR) crowdfunding pilot for LMIC researchers. METHODS: Our global systematic review and qualitative evidence synthesis searched six databases for qualitative data. We used a thematic synthesis approach and assessed our findings using the GRADE-CERQual approach. Building on the review findings, we organised a crowdfunding pilot to support LMIC researchers and use crowdfunding. The pilot provided an opportunity to assess the feasibility of crowdfunding for infectious diseases of poverty research in resource-constrained settings. RESULTS: Nine studies were included in the qualitative evidence synthesis. We identified seven findings which we organised into three broad domains: public engagement strategies, correlates of crowdfunding success and risks and mitigation strategies. Our pilot data suggest that crowdfunding is feasible in diverse LMIC settings. Three researchers launched crowdfunding campaigns, met their goals and received substantial monetary (raising a total of US$26 546 across all three campaigns) and non-monetary contributions. Two researchers are still preparing for the campaign launch due to COVID-19-related difficulties. CONCLUSION: Public engagement provides a foundation for effective crowdfunding for health research. Our evidence synthesis and pilot data provide practical strategies for LMIC researchers to engage the public and use crowdfunding. A practical guide was created to facilitate these activities across multiple settings.

Scaffold-Free Endometrial Organoids Respond to Excess Androgens Associated With Polycystic Ovarian Syndrome.

CONTEXT: Polycystic ovary syndrome (PCOS) is a prevalent disorder in reproductive aged women associated with a number of endocrine and metabolic complications, including increased risk of endometrial cancer. OBJECTIVE: To study the effect of the characteristic increased androgen levels in PCOS on the endometrium, a novel scaffold-free multicellular endometrial organoid was established. DESIGN: Human endometrial organoids were constructed using primary endometrial epithelial and stromal cells from endometrial tissues. Organoids were treated for 14 days with physiologic levels of estradiol and testosterone to mimic a normal follicular phase or PCOS hormone profiles. Organoids were harvested for immunostaining and ribonucleic acid sequencing. SETTING: Academic institution. PATIENTS: Endometrial tissues from 10 premenopausal women undergoing hysterectomy for benign pathologies were obtained following written consent. MAIN OUTCOME MEASURES: Organoid architecture, cell specific markers, functional markers, proliferation, and gene expression were measured. RESULTS: A method to generate scaffold-free endometrial organoids containing epithelial and stromal cells was established. These organoids exhibited distinct organization with epithelial cells lining the outer surface and stromal cells in the center of the organoids. Epithelial cells were polarized, organoids expressed cell type specific and functional markers, as well as androgen, estrogen, and progesterone receptors. Treatment with PCOS hormones increased cell proliferation and dysregulated genes in endometrial organoids. CONCLUSIONS: A new multicellular, scaffold-free endometrial organoid system was established that resembled physiology of the native endometrium. Excess androgens in PCOS promoted cell proliferation in endometrial organoids, revealing new mechanisms of PCOS-associated with risk of endometrial neoplasia.

Generation of Multicellular Human Primary Endometrial Organoids.

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

Author Correction: Trans-differentiation of outer hair cells into inner hair cells in the absence of INSM1.

In Figs. 1e and 2g of this Letter, the labels 'actin' and 'VGLUT3', respectively, should have been in red instead of green font. This has been corrected online.

Trans-differentiation of outer hair cells into inner hair cells in the absence of INSM1.

The mammalian cochlea contains two types of mechanosensory hair cell that have different and critical functions in hearing. Inner hair cells (IHCs), which have an elaborate presynaptic apparatus, signal to cochlear neurons and communicate sound information to the brain. Outer hair cells (OHCs) mechanically amplify sound-induced vibrations, providing enhanced sensitivity to sound and sharp tuning. Cochlear hair cells are solely generated during development, and hair cell death-most often of OHCs-is the most common cause of deafness. OHCs and IHCs, together with supporting cells, originate in embryos from the prosensory region of the otocyst, but how hair cells differentiate into two different types is unknown1-3. Here we show that Insm1, which encodes a zinc finger protein that is transiently expressed in nascent OHCs, consolidates their fate by preventing trans-differentiation into IHCs. In the absence of INSM1, many hair cells that are born as OHCs switch fates to become mature IHCs. To identify the genetic mechanisms by which Insm1 operates, we compared the transcriptomes of immature IHCs and OHCs, and of OHCs with and without INSM1. In OHCs that lack INSM1, a set of genes is upregulated, most of which are normally preferentially expressed by IHCs. The homeotic cell transformation of OHCs without INSM1 into IHCs reveals a mechanism by which these neighbouring mechanosensory cells begin to differ: INSM1 represses a core set of early IHC-enriched genes in embryonic OHCs and makes them unresponsive to an IHC-inducing gradient, so that they proceed to mature as OHCs. Without INSM1, some of the OHCs in which these few IHC-enriched transcripts are upregulated trans-differentiate into IHCs, identifying candidate genes for IHC-specific differentiation.

Codeficiency of Lysosomal Mucolipins 3 and 1 in Cochlear Hair Cells Diminishes Outer Hair Cell Longevity and Accelerates Age-Related Hearing Loss.

Acquired hearing loss is the predominant neurodegenerative condition associated with aging in humans. Although mutations on several genes are known to cause congenital deafness in newborns, few genes have been implicated in age-related hearing loss (ARHL), perhaps because its cause is likely polygenic. Here, we generated mice lacking lysosomal calcium channel mucolipins 3 and 1 and discovered that both male and female mice suffered a polygenic form of hearing loss. Whereas mucolipin 1 is ubiquitously expressed in all cells, mucolipin 3 is expressed in a small subset of cochlear cells, hair cells (HCs) and marginal cells of the stria vascularis, and very few other cell types. Mice lacking both mucolipins 3 and 1, but not either one alone, experienced hearing loss as early as at 1 month of age. The severity of hearing impairment progressed from high to low frequencies and increased with age. Early onset of ARHL in these mice was accompanied by outer HC (OHC) loss. Adult mice conditionally lacking mucolipins in HCs exhibited comparable auditory phenotypes, thereby revealing that the reason for OHC loss is mucolipin codeficiency in the HCs and not in the stria vascularis. Furthermore, we observed that OHCs lacking mucolipins contained abnormally enlarged lysosomes aggregated at the apical region of the cell, whereas other organelles appeared normal. We also demonstrated that these aberrant lysosomes in OHCs lost their membrane integrity through lysosomal membrane permeabilization, a known cause of cellular toxicity that explains why and how OHCs die, leading to premature ARHL.SIGNIFICANCE STATEMENT Presbycusis, or age-related hearing loss (ARHL), is a common characteristic of aging in mammals. Although many genes have been identified to cause deafness from birth in both humans and mice, only a few are known to associate with progressive ARHL, the most prevalent form of deafness. We have found that mice lacking two lysosomal channels, mucolipins 3 and 1, suffer accelerated ARHL due to auditory outer hair cell degeneration, the most common cause of hearing loss and neurodegenerative condition in humans. Lysosomes lacking mucolipins undergo organelle membrane permeabilization and promote cytotoxicity with age, revealing a novel mechanism of outer hair cell degeneration and ARHL. These results underscore the importance of lysosomes in hair cell survival and the maintenance of hearing.

Three members of a peptide family are differentially distributed and elicit differential state-dependent responses in a pattern generator-effector system.

C-type allatostatins (AST-Cs) are pleiotropic neuropeptides that are broadly conserved within arthropods; the presence of three AST-C isoforms, encoded by paralog genes, is common. However, these peptides are hypothesized to act through a single receptor, thereby exerting similar bioactivities within each species. We investigated this hypothesis in the American lobster, Homarus americanus, mapping the distributions of AST-C isoforms within relevant regions of the nervous system and digestive tract, and comparing their modulatory influences on the cardiac neuromuscular system. Immunohistochemistry showed that in the pericardial organ, a neuroendocrine release site, AST-C I and/or III and AST-C II are contained within distinct populations of release terminals. Moreover, AST-C I/III-like immunoreactivity was seen in midgut epithelial endocrine cells and the cardiac ganglion (CG), whereas AST-C II-like immunoreactivity was not seen in these tissues. These data suggest that AST-C I and/or III can modulate the CG both locally and hormonally; AST-C II likely acts on the CG solely as a hormonal modulator. Physiological studies demonstrated that all three AST-C isoforms can exert differential effects, including both increases and decreases, on contraction amplitude and frequency when perfused through the heart. However, in contrast to many state-dependent modulatory changes, the changes in contraction amplitude and frequency elicited by the AST-Cs were not functions of the baseline parameters. The responses to AST-C I and III, neither of which is COOH-terminally amidated, are more similar to one another than they are to the responses elicited by AST-C II, which is COOH-terminally amidated. These results suggest that the three AST-C isoforms are differentially distributed in the lobster nervous system/midgut and can elicit distinct behaviors from the cardiac neuromuscular system, with particular structural features, e.g., COOH-terminal amidation, likely important in determining the effects of the peptides. NEW & NOTEWORTHY Multiple isoforms of many peptides exert similar effects on neural circuits. In this study we show that each of the three isoforms of C-type allatostatin (AST-C) can exert differential effects, including both increases and decreases in contraction amplitude and frequency, on the lobster cardiac neuromuscular system. The distribution of effects elicited by the nonamidated isoforms AST-C I and III are more similar to one another than to the effects of the amidated AST-C II.

Mucolipin co-deficiency causes accelerated endolysosomal vacuolation of enterocytes and failure-to-thrive from birth to weaning.

During the suckling period, intestinal enterocytes are richly endowed with endosomes and lysosomes, which they presumably utilize for the uptake and intracellular digestion of milk proteins. By weaning, mature intestinal enterocytes replace those rich in lysosomes. We found that mouse enterocytes before weaning express high levels of two endolysosomal cation channels, mucolipins 3 and 1 -products of Trpml3 and Trpml1 genes; moreover neonatal enterocytes of mice lacking both mucolipins (Trpml3-/-;Trpml1-/-) vacuolated pathologically within hours of birth and remained so until weaning. Ultrastructurally and chemically these fast-forming vacuoles resembled those that systemically appear in epithelial cells of mucolipidosis type IV (MLIV) patients, which bear mutations in Trpml1. Hence, lack of both mucolipins 1 and 3 causes an accelerated MLIV-type of vacuolation in enterocytes. The vacuoles were aberrant hybrid organelles with both endosomal and lysosomal components, and were not generated by alterations in endocytosis or exocytosis, but likely by an imbalance between fusion of lysosomes and endosomes and their subsequent scission. However, upon extensive vacuolation enterocytes displayed reduced endocytosis from the intestinal lumen, a defect expected to compromise nutrient uptake. Mice lacking both mucolipins suffered a growth delay that began after birth and continued through the suckling period but recovered after weaning, coinciding with the developmental period of enterocyte vacuolation. Our results demonstrate genetic redundancy between lysosomal mucolipins 3 and 1 in neonatal enterocytes. Furthermore, our Trpml3-/-;Trpml1-/- mice represent a polygenic animal model of the poorly-understood, and often intractable, neonatal failure-to-thrive with intestinal pathology. Our results implicate lysosomes in neonatal intestinal pathologies, a major cause of infant mortality worldwide, and suggest transient intestinal dysfunction might affect newborns with lysosomal storage disorders. Finally, we conclude that mucolipin-endowed lysosomes in the young play an evolutionarily-conserved role in the intracellular digestion of maternally-provided nutrients, whether milk in mammals or yolk in oviparous species.

TRPML2 and mucolipin evolution.

The TRPML2 protein, encoded by the Mcoln2 gene, is one of the three mucolipins (TRPML1-3), a subset of the TRP superfamily of ion channels. Although there are no thorough studies on the cellular distribution of TRPML2, its mRNA appears to be largely restricted to lymphocytes and other immune cells. This contrasts with the ubiquitous expression of TRPML1 and the limited but diverse expression of TRPML3 and clearly suggests a specialized role for TRPML2 in immunity. Localization studies indicate that TRPML2 is present in lysosomes (including the specialized lysosome-related organelle that B-lymphocytes use for processing of the antigen-bound B-cell receptor), late endosomes, recycling endosomes, and, at a much lower level, the plasma membrane. Heterologously expressed TRPML2, like TRPML1 and/or TRPML3, forms ion channels that can be activated by a gain-of-function mutation (alanine to proline in the fifth transmembrane domain, close to the pore) that favors the open state, by a transient reduction of extracellular sodium followed by sodium replenishment, by small chemicals related to sulfonamides, and by PI(3,5)P2, a rare phosphoinositide that naturally accumulates in the membranes of endosomes and lysosomes and thus could act as a physiologically relevant agonist. TRPML2 channels are inwardly rectifying and permeable to Ca(2+), Na(+), and Fe(2+). When heterologously co-expressed, TRPML2 can form heteromultimers with TRPML1 and TRPML3. In B-lymphocytes, TRPML2 and TRPML1 may play redundant roles in the function of their specialized lysosome. Although the specific subcellular function of TRPML2 is unknown, distribution and channel properties suggest roles in calcium release from endolysosomes, perhaps to regulate vesicle fusion and/or subsequent scission or to release calcium from intracellular acidic stores for signaling in the cytosol. Alternatively, TRPML2 could function in the plasma membrane, and its abundance in vesicles of the endocytic pathway could simply be due to regulation by endocytosis and exocytosis. The Mcoln2 gene is closely downstream from and in the same orientation as Mcoln3 in the genomes of most jawed vertebrates (from humans to sharks) with the exception of pigs, Xenopus tropicalis, and ray-finned fishes. The close homology of TRPML2 and 3 (closer to each other than to TRPML1) suggests that Mcoln2 and Mcoln3 arose from unequal crossing over that duplicated a common ancestor and placed both gene copies in tandem. These genes would have come apart subsequently in pigs, Xenopus, and the ancestor to ray-finned fishes. All jawed vertebrates for which we have thorough genomic knowledge have distinct Mcoln1, 2, and 3 genes (except ray-finned fishes which, probably due to the whole-genome duplication in their common ancestor, have two Mcoln1-like genes and two Mcoln3-like genes, although only one Mcoln2 gene). However, the available genomes of invertebrate deuterostomes (a sea urchin, lancelet, and two tunicates) contain a single mucolipin gene that is equally distant from the three vertebrate mucolipins. Hence, vertebrate mucolipins arose through two rounds of gene duplication (the first one likely producing Mcoln1 and the ancestor to Mcoln2 and 3) at some time between the onset of craniates and that of jawed vertebrates. This is also the evolutionary period during which adaptive immunity appeared. Given the restricted expression of TRPML2 in immune cells, this evolutionary history suggests a functional role in the adaptive immunity characteristic of vertebrates.

Inter-animal variability in the effects of C-type allatostatin on the cardiac neuromuscular system in the lobster Homarus americanus.

Although the global effects of many modulators on pattern generators are relatively consistent among preparations, modulators can induce different alterations in different preparations. We examined the mechanisms that underlie such variability in the modulatory effects of the peptide C-type allatostatin (C-AST; pQIRYHQCYFNPISCF) on the cardiac neuromuscular system of the lobster Homarus americanus. Perfusion of C-AST through the semi-intact heart consistently decreased the frequency of ongoing contractions. However, the effect of C-AST on contraction amplitude varied between preparations, decreasing in some preparations and increasing in others. To investigate this variable effect, we examined the effects of C-AST both peripherally and centrally. When contractions of the myocardium were elicited by controlled stimuli, C-AST did not alter heart contraction at the periphery (myocardium or neuromuscular junction) in any hearts. However, when applied either to the semi-intact heart or to the cardiac ganglion (CG) isolated from hearts that responded to C-AST with increased contraction force, C-AST increased both motor neuron burst duration and the number of spikes per burst by about 25%. In contrast, CG output was increased only marginally in hearts that responded to C-AST with a decrease in contraction amplitude, suggesting that the decrease in amplitude in those preparations resulted from decreased peripheral facilitation. Our data suggest that the differential effects of a single peptide on the cardiac neuromuscular system are due solely to differential effects of the peptide on the pattern generator; the extent to which the peptide induces increased burst duration is crucial in determining its overall effect on the system.

Identification of SYWKQCAFNAVSCFamide: a broadly conserved crustacean C-type allatostatin-like peptide with both neuromodulatory and cardioactive properties.

The allatostatins comprise three structurally distinct peptide families that regulate juvenile hormone production by the insect corpora allata. A-type family members contain the C-terminal motif -YXFGLamide and have been found in species from numerous arthropod taxa. Members of the B-type family exhibit a -WX(6)Wamide C-terminus and, like the A-type peptides, appear to be broadly conserved within the Arthropoda. By contrast, members of the C-type family, typified by the unblocked C-terminus -PISCF, a pyroglutamine blocked N-terminus, and a disulfide bridge between two internal Cys residues, have only been found in holometabolous insects, i.e. lepidopterans and dipterans. Here, using transcriptomics, we have identified SYWKQCAFNAVSCFamide (disulfide bridging predicted between the two Cys residues), a known honeybee and water flea C-type-like peptide, from the American lobster Homarus americanus (infraorder Astacidea). Using matrix assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS), a mass corresponding to that of SYWKQCAFNAVSCFamide was detected in the H. americanus brain, supporting the existence of this peptide and its theorized structure. Furthermore, SYWKQCAFNAVSCFamide was detected by MALDI-FTMS in neural tissues from five additional astacideans as well as 19 members of four other decapod infraorders (i.e. Achelata, Anomura, Brachyura and Thalassinidea), suggesting that it is a broadly conserved decapod peptide. In H. americanus, SYWKQCAFNAVSCFamide is capable of modulating the output of both the pyloric circuit of the stomatogastric nervous system and the heart. This is the first demonstration of bioactivity for this peptide in any species.