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Microorganisms (sensu lato, i.e., including micrometazoans) are thought to have cosmopolitan geographic distributions due to their theoretically unlimited dispersal capabilities, a consequence of their tiny size, population dynamics, and resistant forms. However, several molecular studies of microorganisms have identified biogeographic patterns indicating cryptic speciation and/or weak species definitions. Using a multi-locus approach with the genus Milnesium (Tardigrada), we aimed to determine the genetic structure of populations worldwide and the effects of long distance dispersal (LDD) on genetic connectivity and relationships across the six continents. Our results on this micrometazoan's genetic structure and LDD at global and micro-local scales indicate contrasting patterns not easily explained by a unique or simple phenomenon. Overall, we report three key findings: (i) confirmation of long distance dispersal for tardigrades, (ii) populations with globally-shared or endemic micro-local haplotypes, and (iii) a supported genetic structure instead of the homogeneous genetic distribution hypothesized for microorganisms with LDD capabilities. Moreover, incongruences between our morphological and molecular results suggest that species delimitation within the genus Milnesium could be problematic due to homoplasy. Duality found for Milnesium populations at the global scale, namely, a molecular phylogenetic structure mixed with widely distributed haplotypes (but without any apparent biogeographic structure), is similar to patterns observed for some unicellular, prokaryotic and eukaryotic, microorganisms. Factors influencing these patterns are discussed within an evolutionary framework.

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Nuclear magnetic resonance (NMR) experiments on subnanoliter (sub-nL) volumes are hindered by the limited sensitivity of the detector and the difficulties in positioning and holding such small samples in proximity of the detector. In this work, we report on NMR experiments on liquid and biological entities immersed in liquids having volumes down to 100 pL. These measurements are enabled by the fabrication of high spatial resolution 3D printed microfluidic structures, specifically conceived to guide and confine sub-nL samples in the sub-nL most sensitive volume of a single-chip integrated NMR probe. The microfluidic structures are fabricated using a two-photon polymerization 3D printing technique having a resolution better than 1 µm3. The high spatial resolution 3D printing approach adopted here allows to rapidly fabricate complex microfluidic structures tailored to position, hold, and feed biological samples, with a design that maximizes the NMR signals amplitude and minimizes the static magnetic field inhomogeneities. The layer separating the sample from the microcoil, crucial to exploit the volume of maximum sensitivity of the detector, has a thickness of 10 µm. To demonstrate the potential of this approach, we report NMR experiments on sub-nL intact biological entities in liquid media, specifically ova of the tardigrade Richtersius coronifer and sections of Caenorhabditis elegans nematodes. We show a sensitivity of 2.5x1013 spins/Hz1/2 on 1H nuclei at 7 T, sufficient to detect 6 pmol of 1H nuclei of endogenous compounds in active volumes down to 100 pL and in a measurement time of 3 hours. Spectral resolutions of 0.01 ppm in liquid samples and of 0.1 ppm in the investigated biological entities are also demonstrated. The obtained results may indicate a route for NMR studies at the single unit level of important biological entities having sub-nL volumes, such as living microscopic organisms and eggs of several mammalians, humans included.

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Single layer centrifugation (SLC) is a useful technique to select porcine spermatozoa for further artificial insemination practices. The aim of this study was to determine possible side-effects related to capacitation due to the process. Semen viability, acrosome integrity and capacitation status were determined through fluorescent probes (SYBR14-PI, FITC-PSA, CTC staining) and Hsp70 immunolocalization and protein tyrosine phosphorylation (by western blotting and immunolocalization) in different groups: control, after SLC with Androcoll (AND), after SLC and washing (AND-Wash) and after SLC, washing and storage for 2h at 17°C with 2.5% of seminal plasma (AND-Wash-SP). Neither viability nor acrosome integrity were impaired by the different treatments; as far as CTC staining, we observed a significant increase (p<0.05) in the capacitation related pattern in AND and AND-Wash, while after exposure for 2h to seminal plasma (AND-Wash-SP group), the increase became less evident; the same trend was observed in Hsp70 immunolocalization for the EL pattern. Neither immunolocalization nor western blotting for tyrosine phosphorylated proteins had an increase in capacitated pattern or in phosphorylation status, except for a 25kDa band that increased in AND and AND-Wash groups and decreased in AND-Wash-SP group. SLC using Androcoll-P induces some capacitation-related changes in boar sperm membrane, as demonstrated by CTC staining and Hsp70 immunolocalization. For protein tyrosine phosphorylation, only a 25kDa protein showed some changes that should be investigated further.

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Anhydrobiosis is a highly stable state of suspended animation in an organism due to its desiccation, which is followed by recovery after rehydration. Changes occurring during drying could damage molecules, including DNA. Using the anhydrobiotic tardigrade Paramacrobiotus richtersi as a model organism, we have evaluated the effects of environmental factors, such as temperature and air humidity level (RH), on the survival of desiccated animals and on the degradation of their DNA. Tardigrades naturally desiccated in leaf litter and tardigrades experimentally desiccated on blotting paper were considered. Replicates were kept at 37 degrees C and at different levels of RH for 21 days. RH values and temperature, as well as time of exposure to these environmental factors, have a negative effect on tardigrade survival and on the time required by animals to recover active life after desiccation. DNA damages (revealed as single strand breaks) occurred only in desiccated tardigrades kept for a long time at high RH values. These results indicate that during the anhydrobiotic state, damages take place and accumulate with time. Two hypotheses can be formulated to explain the results: (i) oxidative damages occur in desiccated specimens of P. richtersi, and (ii) high temperatures and high RH values change the state of the disaccharide trehalose, reducing its protective role.

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Anhydrobiotic organisms are well known for their resistance to extreme environmental conditions while in the inactive, ametabolic, state. This study confirms that this resistance also transfers to treatments with the fumigant methyl bromide. Live specimens of nematodes, rotifers, and tardigrades were found after 70 h of treatment with methyl bromide. Quantitative estimates of survival in the eutardigrade Richtersius coronifer revealed no difference between treated and untreated samples which had been kept dry for a short time. However, R. coronifer from samples collected 11 months before treatment had lower survival compared to samples collected shortly before the treatment. The fact that anhydrobiotic organisms escape treatments with biocides should be considered a serious problem, both for the food industry and from the perspective of human-mediated spread of organisms.

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The therapeutic value of Direct Intraperitoneal Insemination (DIPI) is of particular interest in cases of unexplained infertility and failed AID. The same technique is less likely to be successful where functional or morphological semen abnormalities or multifactorial infertility exist. The Authors report their own experience with DIPI as a treatment on 76 couples with a long-standing infertility.

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Selective cannulation of the Fallopian tubes via the utero-tubal junction, without analgesia, anaesthesia or surgery, has been carried out to replace three embryos in a patient. Cannulating the Fallopian tubes did not appear materially to affect the transport of embryos or the receptivity of the endometrium for implantation.

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In the last decade the strategy for the prevention of the neonatal respiratory distress syndrome (RDS) has been directed towards the acceleration of foetal lung maturation in utero by means of drugs administered to the mother, the most thoroughly investigated being glucocorticoids. Harmful potential side effects of glucocorticoids have led to testing of other drugs capable of accelerating foetal lung maturation, including ambroxol and aminophylline. More recently supplementary surfactant instilled into the trachea has been shown to improve oxygenation of premature babies and to reduce the severity of RDS. To minimise the incidence of neonatal RDS it is important to identify pregnant women at risk for preterm labour and to establish specific guidelines for the use of any prenatal drug to be administered for prevention of RDS. It is also mandatory to assess foetal lung maturity in case of preterm delivery, to monitor the foetus intensively intrapartum, to prevent birth asphyxia and to control, and promptly correct derangements of neonatal homeostasis.

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Ultrasound records were examined retrospectively in the cases of 70 fetuses with open spina bifida diagnosed by ultrasonography at 16-23 weeks' gestation. Biparietal diameter was below the 5th centile for gestation in 61% and head circumference measurements in 26%. The anterior horn of the lateral cerebral ventricle to hemisphere ratio was above the 95th centile in 77%, and ventriculomegaly of the posterior horn of the lateral venricle was observed in 86%. In the 54 fetuses for which pictures were taken at the level of the biparietal diameter a scalloping of the frontal bones (the lemon sign) was seen. In 12 of 21 fetuses for which a suboccipital bregmatic view of the cranium had been obtained, the cerebellar hemispheres were curved anteriorly with simultaneous obliteration of the cisterna magna (the banana sign); in a further 8 cases the cerebellum was not displayed. None of these changes was seen in 100 patients presenting consecutively for routine ultrasound examination in the second trimester.

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