Molecular Characterisation of Faecal Bacterial Assemblages Among Four Species of Syntopic Odonates.

Factors such as host species, phylogeny, diet, and both timing and location of sampling are thought to influence the composition of gut-associated bacteria in insects. In this study, we compared the faecal-associated bacterial taxa for three Coenagrion and one Enallagma damselfly species. We expected high overlap in representation of bacterial taxa due to the shared ecology and diet of these species. Using metabarcoding based on the 16S rRNA gene, we identified 1513 sequence variants, representing distinct bacterial 'taxa'. Intriguingly, the damselfly species showed somewhat different magnitudes of richness of ZOTUs, ranging from 480 to 914 ZOTUs. In total, 921 (or 60.8% of the 1513) distinct ZOTUs were non-shared, each found only in one species, and then most often in only a single individual. There was a surfeit of these non-shared incidental ZOTUs in the Enallagma species accounting for it showing the highest bacterial richness and accounting for a sample-wide pattern of more single-species ZOTUs than expected, based on comparisons to the null model. Future studies should address the extent to which faecal bacteria represent non-incidental gut bacteria and whether abundant and shared taxa are true gut symbionts. Pictures of odonates adopted from Norske Art databank under Creative Commons License (CC BY 4.0).

Compact hematite buffer layer as a promoter of nanorod photoanode performances.

The effect of a thin α-Fe2O3 compact buffer layer (BL) on the photoelectrochemical performances of a bare α-Fe2O3 nanorods photoanode is investigated. The BL is prepared through a simple spray deposition onto a fluorine-doped tin oxide (FTO) conducting glass substrate before the growth of a α-Fe2O3 nanorods via a hydrothermal process. Insertion of the hematite BL between the FTO and the nanorods markedly enhances the generated photocurrent, by limiting undesired losses of photogenerated charges at the FTO||electrolyte interface. The proposed approach warrants a marked improvement of material performances, with no additional thermal treatment and no use/dispersion of rare or toxic species, in agreement with the principles of green chemistry.

Pt-functionalized Fe2O3 photoanodes for solar water splitting: the role of hematite nano-organization and the platinum redox state.

Pt/α-Fe2O3 nanocomposites were synthesized on fluorine-doped tin oxide (FTO) substrates by a sequential plasma enhanced-chemical vapor deposition (PE-CVD)/radio frequency (RF) sputtering approach, tailoring the overall Pt content as a function of sputtering time. The chemico-physical properties of the as-prepared systems were extensively investigated by means of complementary techniques, including X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), field emission-scanning electron microscopy (FE-SEM), energy dispersive X-ray spectroscopy (EDXS), secondary ion mass spectrometry (SIMS), and optical absorption spectroscopy, and compared to those of the homologous Pt/α-Fe2O3 systems annealed in air prior and/or after sputtering. The obtained results evidenced that the material compositional, structural and morphological features, with particular regard to the Pt oxidation state and hematite nano-organization, could be finely tailored as a function of the adopted processing conditions. Pt/α-Fe2O3 systems were finally tested as photoanodes in photoelectrochemical (PEC) water splitting experiments, evidencing a remarkable interplay between functional performances and the above-mentioned material properties, as also testified by transient absorption spectroscopy (TAS) results.

Association between host's genetic diversity and parasite burden in damselflies.

Recent research indicates that low genetic variation in individuals can increase susceptibility to parasite infection, yet evidence from natural invertebrate populations remains scarce. Here, we studied the relationship between genetic heterozygosity, measured as AFLP-based inbreeding coefficient fAFLP , and gregarine parasite burden from eleven damselfly, Calopteryx splendens, populations. We found that in the studied populations, 5-92% of males were parasitized by endoparasitic gregarines (Apicomplexa: Actinocephalidae). Number of parasites ranged from none to 47 parasites per male, and parasites were highly aggregated in a few hosts. Mean individual fAFLP did not differ between populations. Moreover, we found a positive association between individual's inbreeding coefficient and parasite burden. In other words, the more homozygous the individual, the more parasites it harbours. Thus, parasites are likely to pose strong selection pressure against inbreeding and homozygosity. Our results support the heterozygosity-fitness correlation hypothesis, which suggests the importance of heterozygosity for an individual's pathogen resistance.

Directed electron transfer in Langmuir-Schäfer layers of porphyrin-fullerene and phthalocyanine-fullerene dyads in inverted organic solar cells.

In this study double linked porphyrin-fullerene and phthalocyanine-fullerene dyads and a single linked phthalocyanine-fullerene dyad were studied as components in inverted organic solar cells (OSCs) equipped with the well known P3HT:PCBM bulk heterojunction as the photoactive layer. The dyad monolayers were deposited onto a surface of P3HT:PCBM by using the Langmuir-Schäfer method, therefore forming oriented monolayers in which the electron donor (D) and the acceptor (A) exist as a close proximity pair in a 1:1 molar ratio. As a result of this structure short circuit current density (J(sc)), open circuit voltage (V(oc)), and power conversion efficiency (η) increased, while the fill factor (FF) remained the same. The devices which contained dyads with double linkage produced higher efficiencies than the one with a single linked dyad. This result can be explained in terms of molecular orientation. It was also verified that the prepared OSC devices have promising long term air stability.

The identification of secreted carbonic anhydrase VI as a constitutive glycoprotein of human and rat milk.

In addition to essential nutrients, human milk contains several classes of bioactive factors such as enzymes, hormones, and growth factors, many of which are implicated in infantile growth and development. Secretory carbonic anhydrase isoenzyme VI (CA VI) has been identified earlier as an essential component of mammalian saliva, and we demonstrate here by using biochemical and immunohistochemical techniques that it is also an elementary component of milk. The 42-kDa glycopolypeptide purified from human milk in CA inhibitor affinity chromatography shared 100% homology with salivary CA VI in the protein sequence analysis (40% coverage), and its digestion with PNGase F resulted in a polypeptide backbone similar in size to salivary CA VI. Quantification of CA VI in milk by using a time-resolved immunofluorometric assay revealed an approximately eight-times-higher concentration in human colostrum than in mature milk, the latter corresponding to the levels previously detected in human saliva. The high concentration in the colostrum, in particular its functional and structural stability in an acidic milieu, and its growth-supporting role in the taste buds suggest that milk CA VI is an essential factor in normal growth and development of the infant alimentary tract.

An apical membrane Na+/H+ exchanger isoform, NHE-3, is present in the rat epididymal epithelium.

An acidic milieu is required for sperm maturation and for keeping sperm quiescent during storage in the cauda epididymidis. Previous studies have implicated a Na+/H+ exchanger (NHE) in epididymal acidification together with carbonic anhydrase (CA) and vacuolar proton adenosine triphosphatase (H(+)-ATPase). The present studies were undertaken to discover whether the NHE isoform involved is NHE-3, which is known to mediate Na+ and HCO3- absorption in renal tubules. Using the reverse transcription polymerase chain reaction technique (RT-PCR), Northern blot analysis and in situ hybridization, NHE-3 mRNA was detected mainly in the cauda epididymis and to a lesser extent in other regions of the epididymis. Immunohistochemical studies showed that NHE-3 was present in the apical membranes of the epithelial principal cells and confirmed that its expression is strongest in the cauda region, decreasing towards the more proximal regions. Immunoblotting showed a similar expression pattern. These results demonstrate that NHE-3 is expressed in the rat epididymal duct with strongest expression in its cauda region. These findings are thus consistent with the possibility that NHE-3 in the epididymal duct is involved in luminal Na+ and/or HCO3- absorption, as in the renal proximal tubule, and thereby in the regulation of sperm motility and maturation.

Expression of the transmembrane carbonic anhydrases, CA IX and CA XII, in the human male excurrent ducts.

Testicular fluid is concentrated and acidified during its passage through the excurrent ducts. These processes involve bicarbonate absorption, in which carbonic anhydrases are implicated. In this study, the distribution of two transmembrane carbonic anhydrase isozymes (CA IX and CA XII) in the human excurrent ducts was investigated using isozyme-specific antibodies in conjunction with immunohistochemical and immunoblotting techniques. Specific staining for CA XII was present in the basolateral plasma membrane of the epithelial cells in the efferent ducts, predominantly in the non-ciliated cells. In the epididymal duct, CA XII was detected only in sporadic cells, which also contained CA II, thus suggesting that they are apical mitochondria-rich cells. CA IX was also localized to the basolateral plasma membrane of the epithelium in the efferent ducts, but its staining was weaker and less uniform compared to CA XII. No signal for CA IX was detected in the epididymal duct. Western blot analysis from efferent duct samples revealed specific bands for CA IX and CA XII, confirming that the immunohistochemical stainings represent these isozymes. The expression of CA XII and CA IX in the excurrent duct system and co-expression of CA XII with Aquaporin-1 in the same efferent duct epithelial cells suggest their functional involvement in ion transport and concentration processes of testicular fluid.

Secretion of carbonic anhydrase isoenzyme VI (CA VI) from human and rat lingual serous von Ebner's glands.

Salivary carbonic anhydrase VI (CA VI) appears to contribute to taste function by protecting taste receptor cells (TRCs) from apoptosis. The serous von Ebner's glands locating in the posterior tongue deliver their saliva into the bottom of the trenches surrounding the TRC-rich circumvallate and foliate papillae. Because these glands deliver their saliva directly into the immediate vicinity of TRCs, we investigated whether CA VI is secreted by the von Ebner's glands, using immunochemical techniques. The immunohistochemical results showed that CA VI is present in the serous acinar cells, ductal cells, and ductal content of von Ebner's glands and in the demilune and ductal cells plus ductal content of rat lingual mucous glands. More importantly, CA VI was also detected in taste buds and in the taste pores. Western blotting of saliva collected from the orifices of human von Ebner's glands and CAs purified from rat von Ebner's glands confirmed that CA VI is expressed in these glands and secreted to the bottom of the trenches surrounding the circumvallate and foliate papillae. These findings are consistent with the hypothesis that locally secreted CA VI is implicated in the paracrine modulation of taste function and TRC apoptosis. (J Histochem Cytochem 49:657-662, 2001)

Expression of the membrane-associated carbonic anhydrase isozyme XII in the human kidney and renal tumors.

Carbonic anhydrase isozyme XII (CA XII) is a novel membrane-associated protein with a potential role in von Hippel-Lindau carcinogenesis. Although Northern blotting has revealed positive signal for CA XII in normal human kidney, this is the first study to demonstrate its cellular and subcellular localization along the human nephron and collecting duct. Immunohistochemistry with a polyclonal antibody (PAb) raised against truncated CA XII revealed distinct staining in the basolateral plasma membrane of the epithelial cells in the thick ascending limb of Henle and distal convoluted tubules, and in the principal cells of the collecting ducts. A weak basolateral signal was also detected in the epithelium of the proximal convoluted tubules. In addition to the normal kidney specimens, this immunohistochemical study included 31 renal tumors. CA XII showed moderate or strong plasma membrane-associated expression in most oncocytomas and clear-cell carcinomas. The segmental, cellular, and subcellular distribution of CA XII along the human nephron and collecting duct suggests that it may be one of the key enzymes involved in normal renal physiology, particularly in the regulation of water homeostasis. High expression of CA XII in some renal carcinomas may contribute to its role in von Hippel-Lindau carcinogenesis.

Nuclear NonO/p54(nrb) protein is a nonclassical carbonic anhydrase.

The growing carbonic anhydrase (CA) gene family includes 11 enzymatically active isozymes in mammals. Each of them has a characteristic cellular and subcellular distribution pattern. In this report, we demonstrate for the first time a nuclear protein with CA activity. A polypeptide recognized by CA II antibodies was purified from several rat tissues using CA inhibitor affinity chromatography. This polypeptide of apparent 66 kDa mass was characterized using amino acid sequencing and CA activity measurements. It appeared to be identical to nonO/p54(nrb), a previously cloned and characterized RNA and DNA binding nuclear factor. Recombinant nonO generated in baculovirus bound to the CA inhibitor affinity chromatography matrix and revealed detectable CA activity (25 units/mg). Hansson's histochemical staining of rat lymph nodes followed by light and electron microscopy showed nuclear CA activity in lymphocytes, suggesting that the nuclear nonO protein is catalytically active in vivo. These results demonstrate that a previously known transcription factor is a novel, nonclassical CA. Through its CA activity, the nonO may function in the maintenance of pH homeostasis in the nucleus.

Identification of carbonic anhydrase XII as the membrane isozyme expressed in the normal human endometrial epithelium.

Although previous studies demonstrated carbonic anhydrase (CA) activity in the human endometrium, the CA isozyme(s) responsible for this activity has not been established. In this report, we provide the first evidence that the CA isozyme XII, a recently identified transmembrane isozyme that is expressed in normal kidney and greatly overexpressed in some renal cancers, is present in endometrium. We show by immunohistochemistry that CA XII is expressed in the basolateral plasma membrane of epithelial cells of normal human endometrium. Expression of CA XII in uterus was confirmed by Northern blotting. Detergent-solubilized CA XII was isolated from human endometrium by inhibitor affinity chromatography and characterized by isoelectric focusing and Western blot as a polypeptide with a pI of 6.3. The high expression of CA XII in the endometrial epithelium suggests that it may be functionally linked to the pH-dependent events in spermatozoa that precede fertilization. Its basolateral location and extracellular active site could also allow it to influence the morphological changes in endometrium that occur during the menstrual cycle.

Regional expression and androgen regulation of carbonic anhydrase IV and II in the adult rat epididymis.

Carbonic anhydrase (CA) is implicated in the acidification of epididymal fluid and thereby in the regulation of sperm maturation and motility. Among the CA isoenzymes, CA IV and II have been shown to be present in the rat epididymal duct epithelium. In the present study, we examined the expression and androgen regulation of CA IV and II mRNAs along the epididymal duct. Northern blot analysis revealed the presence of CA II mRNA in all regions of the epididymis with the strongest signal in the corpus region, while CA IV mRNA was expressed predominantly in the corpus epididymidis. Three days after bilateral castration, CA IV and II mRNAs were decreased by 80-90% in the corpus epididymidis. Testosterone (T) replacement maintained the expression of CA mRNAs at 50-60% of the control levels, indicating that circulating androgens alone are not sufficient to recover the CA expression in the corpus region. However, unilateral castration did not affect the mRNA levels of CA IV and II, suggesting that factors in testicular fluid do not play a major role in the regulation of CA expression in the corpus epididymidis. Immunoblot analysis showed that CA IV protein levels decreased 3 days after castration, while T administration maintained the protein expression virtually at the precastration levels. These data demonstrate that mRNAs for CA IV and II are predominantly expressed in the corpus region of the rat epididymis and can be regulated by androgens in that region. The present data suggest that the regulation of CA expression in the corpus epididymidis by androgens contributes to the known androgen effects on epididymal acidification.

Membrane-bound carbonic anhydrase IV is expressed in the luminal plasma membrane of the human gallbladder epithelium.

Alkaline hepatic bile is acidified in the gallbladder to prevent calcium precipitation and gallstone formation. Because membrane-bound carbonic anhydrase (CA) isoenzyme IV participates with cytoplasmic CA II in the acidification of urine in the kidney, we studied its expression in different regions of the human biliary tract using immunohistochemical techniques. The enzyme was expressed in the apical plasma membrane of the gallbladder epithelial cells and in the endothelium of the subepithelial capillaries. In the liver, some epithelial cells of the large bile ducts showed positive staining. Its presence in the gallbladder epithelium could be confirmed by Western blotting, which showed a single 35-kd polypeptide band, corresponding in molecular weight to the intact enzyme. The majority of the enzyme was phased to Triton X-114 detergent phase. A small amount of 35-kd polypeptide was also seen in the water phase. Smaller proteolytic fragments of the enzyme were not seen, suggesting that the tissue sample was well preserved. The results show that CA IV is expressed in abundance in the human gallbladder epithelium, where it may participate together with cytoplasmic CA II and ion transporters in acidification of the gallbladder bile via bicarbonate reabsorption.

Expression of carbonic anhydrase isoenzymes IV and II in rat epididymal duct.

Acidic epididymal fluid mainly accounts for sperm quiescence during storage in the epididymis. Carbonic anhydrase (CA) is an enzyme involved in proton and bicarbonate secretion in various epithelia. Therefore, we elucidated the distribution of the cytoplasmic (CA II) and membrane-associated (CA IV) isoenzymes in rat epididymis using polyclonal rabbit antisera to these isoenzymes in conjunction with immunohistochemical and immunoblotting techniques. CA IV was localized in the apical plasma membrane of principal epithelial cells in the distal caput, corpus, and proximal cauda epididymides, the staining intensity being most intense in the corpus segment. The epithelium of the ductus deferens, seminal vesicle, and ventral prostate was devoid of staining. CA II was present in the narrow cells of the initial segment and in the epithelial cells of the distal caput, corpus, and proximal cauda epididymides. Immunoblotting of different epididymal segments for CA IV and II revealed with anti-CA IV serum a distinct 39-kDa polypeptide band in the corpus segment and with anti-CA II serum a 29-kDa polypeptide band in all segments, with the band most intense, however, in the corpus segment. Our results imply that in rat epididymis both bicarbonate reabsorption and proton secretion are involved in epididymal fluid acidification. By analogy with the kidney proximal tubule, we suggest that CA IV is involved in bicarbonate reabsorption mainly occurring in the corpus epididymidis. The presence of CA II in epididymal epithelial cells is probably involved in the supply of protons for secretion mediated by various ion transport mediators.

Location of a membrane-bound carbonic anhydrase isoenzyme (CA IV) in the human male reproductive tract.

We studied the location of a membrane-bound carbonic anhydrase (CA IV) in the human male reproductive tract using a specific antiserum to human CA IV in conjunction with immunoblotting, immunoperoxidase, and immunofluorescence techniques. The microvilli and apical plasma membrane of the epithelial cells and the subepithelial smooth muscle layer of the epididymis, ductus deferens, and ampulla of the ductus deferens showed specific staining for CA IV. The epithelial cells of the prostate and seminal vesicle failed to stain for CA IV, however, whereas the subepithelial smooth muscle layer showed positive staining. No specific staining for CA II was seen in the epithelium of the epididymal duct or the proximal ductus deferens. The presence of CA IV in the epididymis was confirmed by immunoblotting, which revealed 35 KD and 33 KD polypeptides. The results show that the microvilli and the apical plasma membrane of the lining epithelium of the epididymal duct, ductus deferens, and ampulla of the ductus deferens contain the membrane-bound carbonic anhydrase isoenzyme IV. The presence of the enzyme in the epithelium of the epididymis and ductus deferens is probably linked to the acidification of the epididymal fluid that prevents premature sperm activation. Its physiological role in the smooth muscle cells remains to be elucidated.

A high activity carbonic anhydrase isoenzyme (CA II) is present in mammalian spermatozoa.

Human and rat spermatozoa were stained for different carbonic anhydrase (CA) isoenzymes using specific antisera to human CA I, II and VI in conjunction with the immunofluorescence technique. The spermatozoa of both species were found to contain only CA II, which was located principally in the postacrosomal region of the human spermatozoa and in the acrosomal cap region of the rat spermatozoa. The presence of CA II could be confirmed by immunoblotting, which revealed a 29 K polypeptide in both the human and rat spermatozoa. No CA I or VI-specific fluorescence could be detected in the spermatozoa of either species. The immunoblottings were also negative. The results show mammalian spermatozoa to contain the high activity carbonic anhydrase isoenzyme II. Its presence is probably linked to hydration of CO2 produced by active energy metabolism and thereby to the maintaining of an adequate intraspermatozoal bicarbonate concentration as required for the maintenance of sperm motility.

Immunohistochemical localization of carbonic anhydrase isoenzymes VI, II, and I in human parotid and submandibular glands.

Human salivary carbonic anhydrase (HCA VI) was purified by inhibitor affinity chromatography and its location in the human parotid and submandibular glands identified, using a polyclonal antiserum raised against the purified enzyme in rabbits in conjunction with the peroxidase-antiperoxidase complex method. The antibodies raised against the purified enzyme in rabbits did not crossreact with the HCA II or I. However, they slightly recognized human IgA; the antiserum was therefore absorbed with human IgA before immunohistochemical use. HCA VI-specific staining was detected in the cytoplasm and particularly in the secretory granules of the serous acinar cells of both parotid and submandibular glands, the staining of the secretory granules being most distinct in paraformaldehyde-fixed tissues. Some epithelial cells and the luminal content of the striated ducts also gave a specific HCA VI staining. Staining specific for HCA II was also found in the granules of the serous acinar cells, particularly in the submandibular gland when Carnoy fluid fixation was used. Slight HCA II-specific staining was also detected in the striated ductal cells in the Carnoy fluid-fixed specimens. No staining specific for HCA I was detected. The results indicate that the serous acinar cells in human parotid and submandibular glands contain abundant HCA II and HCA VI. Interestingly, only HCA VI is secreted into the saliva, although both enzymes appear to be located in structures resembling the secretory granules in the acinar cells. The enzymes probably form a mutually complementary system regulating the salivary buffer capacity.

Immunohistochemical localization of carbonic anhydrase isoenzymes in the human male reproductive tract.

The distribution of human carbonic anhydrase (HCA) isoenzymes I, II and VI in the human male reproductive tract was studied using specific antisera against affinity purified isoenzymes in conjunction with the peroxidase-antiperoxidase complex method. HCA VI-specific staining could not be demonstrated in any of the tissues studied, and HCA I was observed only in red blood cells. Immunostaining denoted HCA II in the epithelia of the seminal vesicle, ampulla of the ductus deferens and distal ductus deferens. Some cells in the epithelium of the corpus and cauda epididymidis also stained for HCA II. The staining for HCA II in the epithelium of the reproductive tract declined from the strongly positive seminal vesicle to the proximal part of the ductus deferens, which stained negatively. There were also HCA II-positive particles derived from the apical protrusions of the epithelium in the lumina of the seminal vesicle, ampulla of the ductus deferens and ductus deferens. The physiological role of HCA II is linked to the secretion of bicarbonate into the seminal plasma and thereby to the regulation of sperm motility and pH in the seminal plasma.