Mitochondrial DNA (mtDNA) has been used for decades as a predominant tool in population genetics and as a valuable addition to forensic genetic research, owing to its unique maternal inheritance pattern that enables the tracing of individuals along the maternal lineage across numerous generations. The dynamic interplay between evolutionary forces, primarily genetic drift, bottlenecks, and the founder effect, can exert significant influence on genetic profiles. Consequently, the Adriatic islands have accumulated a subset of lineages that exhibits remarkable absence or rarity within other European populations. This distinctive genetic composition underscores the islands’ potential as a significant resource in phylogenetic research, with implications reaching beyond regional boundaries to contribute to a global understanding. In the initial attempt to expand the mitochondrial forensic database of the Croatian population with haplotypes from small isolated communities, we sequenced mitogenomes of rare haplogroups from different Croatian island and mainland populations using next-generation sequencing (NGS). In the next step and based on the obtained results, we refined the global phylogeny of haplogroup N1a, HV2, and X by analyzing rare haplotypes, which are absent from the current phylogenetic tree. The trees were based on 16 novel and 52 previously published samples, revealing completely novel branches in the X and HV2 haplogroups and a new European cluster in the ancestral N1a variant, previously believed to be an exclusively African–Asian haplogroup. The research emphasizes the importance of investigating geographically isolated populations and their unique characteristics within a global context. Isolation, whether geographical or cultural, has always had an impact on the population genetic structure, and it is mirrored in the reduction of genetic diversity due to genetic drift and a higher rate of marriage in consanguinity due to the limited relation options . These fhttps://empop.online/, accessed on 28 June 2023). However, the number of complete mitogenomes in EMPOP (around 4000) is still insufficient and lacks contributions from Croatia [Mitochondrial DNA is also extensively used in forensic casework to characterize biological evidence based on its specific features, such as high copy numbers within cells and the small size of the mitochondrial genome, also very useful in analysis of ancient human remains. The currently available forensic mtDNA database is the EMPOP database, specifically developed for forensic applications, and comprising around 48,500 quality controlled mitotypes  ,23 by usThe initial complete sequencing of 9 mitochondrial genomes was performed at the Estonian Biocentre, Institute of Genomics, Tartu, Estonia using Sanger sequencing. Sequencing was performed on the Applied Biosystems 3730xl DNA Analyzer, Thermo Fischer Scientific, Waltham, MA, USA) with the BigDye Terminator sequencing kit , according to the previously published methodology ,25.n = 32) were performed at the Forensic Science Centre “Ivan Vučetić”, Zagreb, Croatia according to Illumina 2016 Protocol, with the modifications described previously [® GXL DNA polymerase in two amplicons for each sample. The conditions of the optimized protocol for both fragments were as follows: 9.1 kb fragment: 25× ; and 11.2 kb fragment: 25× . Both mtDNA amplicons were then quantified on a QubitTM 3.0 Fluorometer (Thermo Fisher Scientific) with the QubitTM dsDNA High Sensitivity kit. Both mtDNA amplicons were normalized to equal concentrations of 0.2 ng/μL, as described in the Illumina® protocol, and pooled for each sample, wherefrom the volume of 5 μL was taken for library preparation . Nextera® XT Library Prep Kit was used according to the Illumina® protocol, and negative controls (reagent blanks) were used in the target enrichment (NC-PCR) and library preparation (NC-LIB) procedures [® DNA High Sensitivity Assay on LabChip® GX Touch HT  and were diluted to concentrations of 0.2 ng/μL. The libraries normalized to 4 nM were pooled, denatured, and diluted to 12 pM, with 5% PhiX Sequencing Control v. 3  spike-in (Illumina® Denature and Dilute Libraries Guide for MiSeq System). The paired-end sequencing of all 32 samples was performed within a single run on an Illumina® MiSeq FGxTM instrument by using the MiSeq® Reagent Kit v.2, 300 cycles (2 × 151 bp). The run quality metrics were reviewed in Illumina® Sequencing Analysis Viewer (SAV) v.1.11.1 software: a cluster density of 1494 K/mm2, with 87.2% of clusters passing the filter was reported, and 78.8% of the sequenced bases exhibited a base call quality score (Q) above 30.Target enrichment, library preparation, and sequencing of the selected subsample  were mapped to the rCRS (NC_012920) [The raw fastq reads generated by the MiSeq_012920) ,22 using_012920)  was used_012920) . LeftAli_012920) , with a _012920) ,34. The _012920) . Computa_012920) .Coalescence time estimates were computed with the Bayesian MCMC approach implemented in the BEAST v1.7.5 suite of software . All anahttps://sites.google.com/site/mtphyl/home, accessed on 23 February 2023), and the tree topologies were subsequently manually verified, by using all publicly available sequences from the current literature and Mitomap up to March 2023. The trees of the complete mtDNA sequences (52 published and 16 novel ones—9 from our database + 7 sequences from the NGS dataset) were rooted to the Revised Cambridge Reference Sequence (rCRS), in order to enable comparison to the previously published data.The maximum parsimony phylogenetic tree was first constructed with the software mtPhyl 5.003 . The second cluster marked by a 4721 transition encompassed eight different complete sequences from various European regions—four samples of Russian, Greek, and Serbian origin ,11,49, j. The secIn the attempt to trace the geographical distribution of the contemporary N1a variants on a global scale and to revise the generally accepted postulates of their spatial predominance, we created world distribution map for both the N1a 16147A and 16147G variant (depicted in We also reconstructed the phylogenetic tree of the X1’3 using twelve published ,51,52,53The sequencing of two HV2 samples from the Cres Island was also performed and joined with a sample of Armenian origin from the Estonian database, sharing a similar HVSI motif. In the attempt to clarify their unusual connection, we constructed the maximum parsimony phylogenetic tree using eight complete mtDNA sequences from the literature and Mitomap ,56,57,58The Croatian Adriatic islands serve as genetic isolates characterized by the presence of atypical lineages when compared to the contemporary European population, which are more prevalent and frequent due to the influence of genetic drift and recurrent bottlenecks. Incorporating these rare mitogenomes into the EMPOP database would enhance its forensic power by facilitating the identification and matching of individuals possessing uncommon genetic profiles for a particular region. Furthermore, the identification of novel branches within certain haplogroups, such as the HV2, X1’3, and N1a1a ancestral variant, underlines the importance of exploring isolated populations and their distinctive characteristics in shaping the contemporary structure of the human population. Therefore, the objective of this study was to reconstruct the phylogenetic trees for these haplogroups by sequencing rare mitochondrial haplotypes obtained from our database. This research provides valuable insights into the genetic diversity and evolutionary history of these specific lineages, contributing to our understanding of the broader human population structure.The west-Eurasian mitochondrial haplogroup diversity encompasses haplogroups mainly derived from haplogroup R, the daughter branch of the macrohaplogroup N. The estimated origin of haplogroup N was most likely in the Arabian Peninsula around 55–65 kya, shortly after the Out-of-Africa migration, where the derivation into the R clade took place around 59 kya . West EuThe African/South Asian branch, characterized by the 16147G mutation, was believed to be more common in the Arabian Peninsula, northern Africa, and the Near Eastern region and very rare, or even absent in Europe ,67,68. IHaplogroup X was estimated to originate in the Near East ca. 30 kya. According to the current nomenclature it is divided into four subclades denoted as X1–X4. The X2 subclade encompasses by far the largest part of the X tree, marked by both the highest frequency and diversity of all X lineages scattered worldwide It is geographically distributed among West Eurasians, northern groups of Native Americans, as well as in northern Africa and the Near East, but with low frequency, around 1–2% ,16. SomeSimilar interesting trace of a long-distance migration from Near/Middle East in the Adriatic gene pool was found in the HV2 haplogroup. Haplogroup HV is predominantly present in the Near East, Middle East, and in the Caucasus, while in Europe it is spread unevenly—it is rare or absent in the north and west, but more common among southern and eastern Europeans . A revisThe identification of a previously unknown European cluster within the African/South Asian N1a 16147G branch suggests the emergence of a new founder lineage likely originating locally within European territories, potentially predating the Neolithic period. Additionally, the wider distribution of N1a 16147A than previously reported in the literature suggests a need for a revision of the complete N1a phylogeny. Furthermore, the detection of rare mitochondrial lineages, such as X* and HV2b, indicates the presence of signals with origins in the Near/Middle East within the Adriatic gene pool. These findings highlight that small human genetic isolates can serve as reservoirs of population variability, offering insights into prehistoric migratory events that have played a significant role in shaping the maternal genetic landscape on a larger scale.


Sargassum algae, recovered from raw beach seaweed wastes, as a valid source of valuable sodium alginate. Alginate is a biodegradable, highly attractive polysaccharide widely used in food, pharmaceuticals, and biomedicine applications. The aim of this work is to employ a new eco-sustainable and cost-effective extractive method to obtain alginate as a raw material from pollutant organic Sargassum seaweeds. Algae were exposed to microwave pre-treatment under static and dynamic conditions, and three different extractive protocols were followed: (a) conventional, (b) hot water and (c) alkaline method. All samples were characterized by GPC, SEM, FTIR/ATR and TGA. It was found that alginate’s best performances were obtained by the microwave dynamic pre-treatment method followed by alkaline extractive protocol. Nevertheless, the microwave pre-treatment of algae allowed the easiest breaking of their cell walls and the following fast releasing of sodium alginate. The authors demonstrated that microwave-enhanced extraction is an effective way to obtain sodium alginate from Sargassum-stranded seaweed waste materials in a cost-effective and eco-sustainable approach. They also assessed their applications as mulching films for agricultural applications.This paper highlights the potential of  Sargassum algae are brown free-floating seaweed belonging to the Phaeophycean family. They can be found worldwide in maritime temperate and tropical regions, providing refuge, shelter, and food for many animal species, such as sea turtles and shrimps. In recent years, their wide diffusion ashore has gone out of control, even to the point of forming dense clumps of slowly beach-fouling rotting weeds, yielding toxic solid waste alongside urban beaches. In the United States, beach fouling has become a major problem in some urban areas, particularly along the Gulf Coast. Recently, the territory of the Virgin Islands declared a state of emergency due to an unprecedented bloom of seaweed along its coast, which caused widespread beach-fouling and posed a serious risk to public health [c health . Indeed,c health ,3. Neverc health ,5.In the cell tissue, alginates are mainly found as insoluble gels of mixed calcium, magnesium, sodium and potassium salts; they are extracted from the grounded thallium upon the collapse and subsequent transformation of their tissue into a brown mass . ActuallSargassum raw seaweed waste and to use it as a biopolymer matrix for agricultural mulching films. Finalized to this specific aim, sodium alginate extraction and purification do not need to be pushed up to the extremely high level of purity required by pharmaceutical standards. Indeed, for agricultural applications, the retaining of protein, cellulose fractions, fibers and colored pigments, represents an added value in terms of agronomic and mechanical performances of the mulching films on the soil [The purpose of this work is to employ new eco-sustainable and cost-effective extractive methods to obtain alginate from the soil .Ascophyllum nodosum [Nizimuddinia zanardini [Saccharina latissima [Sargassum algae. In fact, MAE has been investigated for the isolation of polyphenols [Sargassum algae, but to the best of our knowledge there is only one report of MAE application to extract alginate from this algae family. In this particular paper, the power level  and microwave exposure time (between 15 and 18 min) were optimized for the extraction of alginate [Sargassum algae wastes, have been used in order to extract sodium alginate by both the conventional protocol and a mild MAE method, in which power density and duration were optimized. In order to enhance the exposure to microwave radiation and minimize the exposure time, a specially designed laboratory-scale fluidized bed was used in the microwave cavity. Fluidized bed technology is long known for its favorable features leading to proper uniformity treatment [When dealing with sodium alginate extractive methods, conventional protocols involve the use of aggressive organic chemicals for the removal of polyphenols and bleaching, large volumes of hot water, dilute acid, dilute alkali and long extraction times. As a consequence, final alginate properties are strongly influenced by the use of aggressive extractive methods, sometimes leading to polymer chemical degradation ,10. Ther nodosum ,14, Nizianardini  and Saccatissima ) and isoyphenols ,18 and fyphenols ,20 from alginate . Framed reatment ,23. In tSargassum algae were kindly supplied from Lianyungang Zhongda Seaweed Industrial Co., LTD . The seaweed was rinsed thoroughly in fresh water to remove sediments and surface contamination. The seaweed was dried in an oven under vacuum at 40 °C for 24 h and stored in a refrigerator at 4 °C until further use. The chemicals used in this study are hydrochloric acid, sodium carbonate, absolute ethanol, acetone  and commercial alginate .The microwave exposure experiments were conducted in triplicates in a laboratory prototype, allowing for the enforcement of optimal power density, duration, thermal regime of working air and exposure uniformity ,25. To oTf. The data were acquired by a service computer. The process cavity had a net capacity of 0.022 m3 and was provided by a MW waveguide and magnetron group  producing a fixed MW power level of 991 W, whose measurement was ensured by using a power clamp meter . To avoid air and vapor pressure build-up in the cavity proper, an exhaust balancing fan was provided to the lateral grille, normally intended to vapor discharge, while the grille on the magnetron side was sealed up. The exhaust fan also provided the air flow seeping through the interspace between the external side of the cavity wall and the cavity’s external case, in order to avoid overheating of the magnetron group. Different MW power levels were provided by insertion of carefully weighted buffer water quantities, placed in the cavity. Different working times were measured by means of a service stopwatch that drove the ON/OFF switch of the magnetron power supply. The effective MW exposure time is set by imposing a magnetron working duration ΔtON, followed by a resting or relaxation duration ΔtOFF. 109 Several “cold” tests  were conducted to ascertain the optimal fluid regime for proper fluidization, hence, to offer a uniform exposure to microwaves.The distributor plate at the bottom of the reactor, and the reactor’s top lid (which included proper apertures for exhaust air) were held in place by Teflon flanges. The reactor was held in place by a rigid wired pedestal, and an optical fiber thermometer fed by a signal conditioner  was inserted at the reactor bottom, to read the fluidization temperature Sargassum particles (particle size between 50–80 mm) underwent three different polysaccharide extraction methods. The first one was performed following the traditional protocol, involving a demineralization process in dilute acid solutions (0.1 M HCl) for 24 h at room temperature and speed of 200 rpm, and following polymer solubilization in alkaline medium (3 wt% Na2CO3) for 3 h at 90 °C and speed of 200 rpm. The second and third ones consisted of a previous exposure of dried chopped substrate to controlled microwave energy in a dedicated prototype, allowing to enforce optimal power density and duration under static and dynamic conditions [tON/ΔtOFF set up 10/50 s. The dynamic conditions correlated to the microwave procedure under the same operative conditions, but this time algae in a wet state, i.e., algae immersed in water, were exposed to the microwave chamber. The mass flow rate of biomass ranged between 0.6 and 0.8 g/min. The pre-treated raw algae particles were then subjected to alginate extraction by using three different routes: (a) hot water treatment for 3 h at 90 °C and 200 rpm; (b) conventional method as described above and (c) alkaline hot treatment (3 wt% Na2CO3) for 3 h at 90 °C and 200 rpm. Following extraction methodologies, the solutions were precipitated in absolute ethanol, washed once in acetone for 30 min and dried. All extracts were further dissolved in water at a constant concentration (1 wt%), poured on Petri dishes, and allowed to dry, in order to obtain alginate-based films.Dried chopped nditions . Static The sodium alginate yield (%) obtained in this research plan is calculated using the following equation:Morphological analysis of raw and treated substrate samples was performed by means of an FEI Quanta 200 FEG Scanning Electron Microscope (SEM) . SEM analyses were performed at room temperature, in high vacuum mode, using a large field detector (LFD) and an acceleration voltage of 20 kV. Prior to the observation, the sample surfaces were coated with a homogeneous layer (18 ± 0.2 nm) of Au-Pd alloy by means of a sputtering device .3 and 0.1 M NaNO3 to avoid any polymer agglomeration phenomena. After complete dissolution, samples were filtered through a 0.22 μm CA filter. The injection volume was 100 μL and the flow rate was 0.5 mL/min. The measurements were performed in duplicate at 40 °C, according to the temperatures of the columns and detectors.Gel permeation chromatography was performed using a GPC Max Viscotek equipped with a TDA 305 composed of refractive index (RI), low angle light scattering (LALS), right angle laser light scattering (RALS), and viscometer (IV) detectors. The column set consisted of a pre-column TSK PWXL and TSK Gel GMPWXL. All the samples were dissolved up to a concentration of ≅0.5–0.7 mg/mL and eluted in MilliQ water containing 0.2% NaN−1, recorded as an average of 16 scans with a resolution of 4 cm−1. Before testing, all samples were dried in an oven at 60 °C for 24 h.Attenuated Total Reflection Fourier Transform Infrared (FTIR-ATR) spectroscopy was carried out on dried films. The spectra were collected on a Perkin–Elmer Spectrum 100 spectrometer , equipped with a Universal ATR diamond optical crystal and ZnSe focusing elements sampling accessory. All the samples were analyzed at room temperature in the range of 4000–650 cmThermogravimetric analyses (TG) were performed by using a thermogravimetric analyzer Mettler-Toledo TG-SDTA 851 thermobalance, equipped with a differential thermal analyzer instrument. About 5 mg of samples were placed in an open ceramic crucible and heated from 25 °C up to 600 °C at a speed rate of 10 °C/min, under a nitrogen flow of 30 mL/min. Before the tests, a blank curve was measured and subtracted from the single thermograms, to correct instrumental drift .Ascophyllum nodosum [Sargassum algae (13–15%) [The different extraction and pre-treatment methods of alginates with sample codes and obtained alginate yields are presented in  nodosum  and for  nodosum , but hig(13–15%) ,34.The micrographs of raw (a) and microwave-treated algae surfaces under static (b) and dynamic (c) conditions are shown in In The molecular weight distributions of all extracted alginates are presented in Sargassum algae by ultrasound-assisted extraction followed by a demineralization process in lemon juice and an alkaline process in Na2CO3 (80–112 kDa) [Sargassum algae [Sargassum algae by alkaline method varying operative parameters (range between 1 and 6 dL/g) [The data in the literature show a broad spectrum of Mw and η values for extracted alginate, which is probably the consequence of different algae species, different extraction methods and extraction parameters. The values of Mw obtained in this work are higher than for alginate produced from 112 kDa) . On the um algae . The vis 6 dL/g) ,43. On t 6 dL/g) ,45.Nevertheless, all extracted alginates in this work have higher Mw than commercial alginate, whereas all samples except hot water treated samples and A3 sample have higher viscosity, suggesting that alternative extraction protocols with reduced amounts of solvents and energy can provide alginate of high quality.−1) and symmetric (1400–1410 cm−1) stretching vibration of carboxylate O–C–O sodium alginate group can be detected in the spectra of all extracted alginates [−1 related to C-C, C-O and C-O-C stretching vibrations of the pyranose ring are mostly marked in A4 and A7 samples.The FTIR/ATR spectra of extracted alginates by MAE under dynamic conditions are presented in lginates ,46. The −1. Generally, alginate displays two peaks in this region, one around 810 cm−1 and one as a small shoulder peak around 780 cm−1 corresponding to mannuronic and guluronic acid, respectively [−1 and shoulder peak at 890 cm−1 are found in the FTIR spectrum of commercial alginate, which is ascribed to the C-O stretching vibration of uronic acid and of C1–H deformation vibration of mannuronic acid, respectively [−1 is detected in all spectra of extracted alginates, and an additional peak of small intensity at 905 cm−1 related to the α-l-guluronic asymmetric ring vibration can be seen, too [−1 in spectra of all extracted alginates, except in the case of A2 and A5 (hot water extraction). Although at that IR frequency is usually C1-H deformation vibration of β-mannuronic acid, the change of peak shape from wide shoulder to narrow peak, and peak intensity from weak to strong, indicates that this is a new peak related to another functional group in alginate. It is found in the literature that peaks at 870–880 cm−1 are typical of carbonate ions vibrations [A particularly interesting area for analysis of polysaccharides is the anomeric region between 950 and 700 cmectively ; these pectively ,49,50. Tbrations ,52. Henc−1 overlapping with the carboxylate asymmetric vibrational peak and another broad stretching mode at 1550 cm−1, corresponding to amide I and amide II protein vibrations, respectively. Finally, a broad peak at 1250 cm−1 was ascribable to the asymmetric stretching vibration of the S=O group [Regarding the hot water extraction, the corresponding samples, A2 and A5, evidence two stretching vibrational groups not detected in other samples; specifically, it is possible to note a broad absorption peak around 1650 cm=O group ,53. ThisTGA analysis of extracted alginates was performed, and their thermal behavior was compared with the one of commercial alginate. Considering the very low sodium alginate extraction yield of A2 and A5 samples and their complex structure, these samples have not been considered in this analysis. In order to better visualize the thermal degradation pattern and kinetics of selected samples, only DTG thermograms are shown , whereasSargassum-derived alginates is shifted to lower values ranging between 27 and 35 °C, if compared with commercial alginate. The highest shift is detected for all samples extracted by the conventional method. It is interesting to note that DTG diagrams of A4 and A7 samples display a wide shoulder peak in the region between 180 and 330 °C, whereas for the A3 and A6 samples doublet peak appears and is probably related to the degradation of mannuronic residues at lower temperature, which is subsequently followed by degradation of guluronic units. This result confirms that microwave pre-treatment combined with the conventional extractive method causes partial hydrolysis of alginate. The double peak during the thermal degradation of extracted alginate by the acid+alkaline method is supported by literature data [onset temperature is significantly lower for all extracts, the maximum thermal degradation of alginate macromolecular chains is delayed by 10 °C, in comparison to the commercial alginate. In addition, there are no significant differences  in degradation pattern between two used microwave pre-treatments samples. The only visible difference is that weight loss rate for the A6 and A7 (microwave dynamic conditions) is lower than for the A3 and A4 (microwave static conditions), which is evidenced in reduced peak intensity in the region between 180 and 330 °C. This result matches well with spectroscopic (FTIR-ATR) and molecular (GPC) analysis.The thermal degradation of commercial alginate occurs in two main steps: the first one up to 100 °C corresponds to the evaporation of free water, and the second step, starting at 215 °C and peaking at 235 °C, is related to the decomposition process of alginate, which involves a random split of the glycosidic bonds, vaporization and elimination of volatile products . All exture data . AlthougSargassum seaweed wastes, preliminary tests have been performed using them as mulching geo-membrane on Hortensia Dienamann (HD) plant. Mulching polysaccharide-based films have gained increased attention in agriculture to minimize weed appearance [In order to evaluate the potential applications of alginate extracted from pearance . Namely,pearance ,60,61, gpearance ,63, and pearance  have proSargassum algae under static and dynamic conditions using a fluidized bed in a microwave cavity was reported. It was demonstrated that sodium alginate can be successfully extracted after both microwave pre-treatment regimes, using mild extracting conditions. The yield of obtained alginates was in the range between 20 and 36%, depending on extraction solvents. The highest yields were obtained when the conventional protocol was applied , and microwave pre-treatment was followed by the acid+alkaline protocol. The microwave pre-treatment method under dynamic conditions was the most efficient approach to obtain alginates of higher molecular weights (419–458 kDa), as opposed to the conventional methods of untreated algae (382 kDa). Although microwave dynamic treatment followed by alkaline extraction produced lower yields of alginates (20–24%) in comparison to the conventional protocol of untreated algae, this method is more time and energy-efficient , requiring less amount of solvents to be used for extraction, and providing the alginates of higher molecular weight and higher thermal stability. Overall, results related to sodium alginate extraction from wastes of raw Sargassum algae, evidenced that controlled microwave treatment under dynamic conditions could really represent a valid method to obtain sodium alginate in a cost-effective and eco-sustainable method, by providing, at the same time, an environmentally friendly approach finalized to the recovering, collection and upgrading of urban beach raw waste materials.In this investigation, microwave-assisted extraction of alginate from


Hydropower dams are a source of renewable energy, but dam development and hydropower generation negatively affect freshwater ecosystems, biodiversity, and food security. We assess the effects of hydropower dam development on spatial–temporal changes in fish biodiversity from 2007 to 2014 in the Sekong, Sesan, and Srepok Basins—major tributaries to the Mekong River. By analyzing a 7-year fish monitoring dataset, and regressing fish abundance and biodiversity trends against cumulative number of upstream dams, we found that hydropower dams reduced fish biodiversity, including migratory, IUCN threatened and indicator species in the Sesan and Srepok Basins where most dams have been constructed. Meanwhile, fish biodiversity increased in the Sekong, the basin with the fewest dams. Fish fauna in the Sesan and Srepok Basins decreased from 60 and 29 species in 2007 to 42 and 25 species in 2014, respectively; while they increased from 33 in 2007 to 56 species in 2014 in the Sekong Basin. This is one of the first empirical studies to show reduced diversity following dam construction and fragmentation, and increased diversity in less regulated rivers in the Mekong River. Our results underscore the importance of the Sekong Basin to fish biodiversity and highlight the likely significance of all remaining free-flowing sections of the Lower Mekong Basin, including the Sekong, Cambodian Mekong, and Tonle Sap Rivers to migratory and threatened fish species. To preserve biodiversity, developing alternative renewable sources of energy or re-operating existing dams to increase power generation are recommended over constructing new hydropower dams. The LMB is also home to a rich diversity of other aquatic fauna such as over 400 species of micro-invertebrates2, and at least 300 macroinvertebrate species such as molluscs3, crustaceans, annelids and aquatic insects6. In the LMB, fish are instrumental to the economy and provide food security for local people8. An estimated 1.3–2.7 million tonnes of fish are harvested from the LMB, valued at ~ $11 billion US dollars (USD) per year in 2015, or approximately $12.9 billion when adjusted for inflation in 20228. However, fish biodiversity and fisheries productivity in the LMB are threatened by hydropower development. Dams disrupt and block fish migrations and alter fish communities and aquatic ecosystems, leading to decreased fish biodiversity, abundance, and biomass9. The decrease in fish yield is estimated to reduce biomass by 725,000 tonnes per year10, affecting food security, biodiversity, and ecosystem services in the Mekong11. This biomass reduction corresponds to an annual GDP loss of about $326 million USD in Cambodia and $165 million USD in Vietnam12.The Mekong River (hereafter the Mekong) is one of the world’s most important biodiversity hotspots. It supports ~ 1200 fish species13. Moreover, 31 additional hydropower dams are under construction, including three on the mainstem Mekong River in Laos. Another 19 large dams (> 200 MW) have been proposed across the LMB. By 2040, estimated economic gain from all projects in the LMB will exceed $160 billion USD, while their potential cost is about $170 billion USD due to declining fisheries, forests, wetlands and mangroves14. Overall, a net economic loss is anticipated from hydropower projects, with the vast majority of benefits accruing to foreign companies and the majority of losses borne by residents of Cambodia, Thailand, and Vietnam, especially rural subsistence fishers who depend on fisheries for food, protein, and livelihoods10.Hydropower dam development projects are booming in the Mekong because increasing energy production is important for the region development. In the LMB, there are 129 commissioned dams, including 5 mainstem Mekong River dams in Laos. All told, 69 projects are located in Laos, 46 in Vietnam, 10 in Thailand, and 4 in Cambodia, which together have capacity to generate 13,340 megawatts (MW) of hydropower7 estimated fish biomass and biodiversity losses based on future dam alternatives and estimated trade-offs between power production and impacts to fish biodiversity. Kano et al.15 modeled the future distribution of fish with intense hydropower development and found reduced species richness and available habitat. Ngor et al.9 assessed the impacts of flow alteration on fish diversity, and reported reductions in local fish richness and abundance in the Sekong, Sesan and Srepok Basins (3S Basin) and altered flow seasonality in the 3S-Mekong-Tonle Sap system, with distinct variations in fish assemblages16. Yoshida et al.12 reviewed the impacts of mainstem hydropower dams on agriculture and fisheries resources, and found that annual fish catch has declined by 276,847 and 178,169 tonnes in Cambodia and Vietnam, respectively. Baird and Green17 and Green and Baird18 reviewed and discussed the impacts of dams on fish and ecosystems and provided options for future clean development. Soukhaphon et al.19 studied several Mekong hydropower dams and found that social and environmental impacts are often cumulative and occur over large scales. All of these studies highlight the significant and varied costs from hydropower development in the LMB and together suggest that further dam construction is not a sustainable development trajectory.The impact of dams on the Mekong’s fish and fisheries have been investigated and summarized in several recent studies. Ziv et al.13. Despite the widespread impacts of hydropower dam development, limited research has quantified spatial and temporal changes in fish biodiversity with dam development.One of the hotspots for hydropower dam development is the Sekong, Sesan and Srepok Basins (3S Basin), which together form a major tributary to the Mekong. In 2007, the Sekong Basin had two dams, the Srepok Basin had two dams, and the Sesan Basin had six dams. In 2014, the number increased to six dams in the Sekong Basin, 13 dams in the Srepok Basin, and 19 dams in the Sesan Basin. Of those dams, six were major dams located on the mainstem river of the Sesan Basin, five were located on the mainstem of Srepok Basin, and one dam was located on the mainstem river of the Sekong Basin—although it was situated far upstream  and Endangered Mekong River Catfish . While the Mekong is home to at least 150 endemic aquatic fauna23, 103 of which are endemic fish species living in the LMB24, it also has approximately 20 non-native species25, which compete with native species for resources24.The 3S Basin has vast ecological importance, with a seasonal flood-pulse that fuels exceptional aquatic biodiversity. Moreover, the 3S Basin is one of the main fish migration routes in the LMB, providing spawning and rearing habitats for some speciesHere, we assess spatial–temporal changes in fish biodiversity from hydropower dam development in the 3S Basin from 2007 to 2014. By using long-term monitoring data, we (1) quantify changes in fish biodiversity composition and indicator species in the 3S Basin, (2) investigate the temporal trends in fish biodiversity  and (3) quantify the impacts of dam development on fish biodiversity. We conclude with discussion about sustainable development futures for the 3S Basin.Our study area had 247 fish species belonging to 43 families and 14 orders. The most common order in the 3S Basin was Cypriniformes , followed by Siluriformes , and Perciformes . The remaining 11 orders made up less than 3% of the total species counts. PCA analysis demonstrated distinct fish communities among the 3S rivers , whereas Srepok Basin had 10 indicator species, including 6 migratory and one Critically Endangered species (Probarbus jullieni). Sesan Basin had only one indicator species (Acantopsis sp.). Fish communities in Sekong Basin were relatively similar between seasons, as indicated by the PCA ordination map , species richness, abundance by fish category  between the river basins Fig. b–d. The In the 3S Basin as a whole, 8 LMB-endemic fish species were observed, with seven, eight, and four species observed in the Sekong, Srepok and Sesan Basins, respectively. Four non-native species were detected throughout the 3S Basin. The Sekong and Srepok Basins each had three non-native species, while there were none in the Sesan Basin over the study period. Refer to Table Overall, fish biodiversity metrics are increasing in the Sekong Basin and declining in the Srepok and Sesan Basins. Linear regression models indicated that fish biodiversity metrics  in the Sekong Basin significantly increased from 2007 to 2014 for total fish and migratory fish. Threatened fish biodiversity metrics showed an upward trend for the Sekong Basin, but only diversity was significant Fig. . In the 2) ranged from 0.06 to 0.42 . Abundance, richness, and diversity always significantly increased in the Sekong Basin for total species and migratory species, with R2 values ranging from 0.47 to 0.79. In contrast, biodiversity trends declined in the Srepok and Sesan Basins as more dams were built ; while in Sesan Basin, total fish abundance and diversity, and the migratory fish diversity significantly decreased  , was an indicator species in the Sekong Basin because they were caught only in this basin and were abundant  in the Sekong Basin, highlighting the basin’s importance to migratory and threatened fish30.Our findings highlight the importance of the Sekong Basin as a free-flowing river corridor for fish migration and suggest that the Sekong Basin potentially compensates for environmental changes and habitat loss in the Sesan and Srepok Basins. The Sekong Basin had the highest number of total, migratory, and threatened fish species observed in the 3S Basin  and the Lower Sekong A (LSKA) dams , provisioning (e.g. water supply and fisheries), and supporting services . Seasonal agricultural activities can also benefit from the free-flowing Sekong because connected rivers ensure seasonal flow availability and support sediment transfer to healthy floodplains51, which all are necessary for agricultural production.Given the negative impacts of hydropower dams on streamflow and water qualityams Fig. a, which ams Fig. a, would 42. Similarly, operating multiple dams as a system, rather than operating dams independently, could optimize hydropower generation and reduce the need for new dams52. In the 3S Basin, 51 dams are already operational, and therefore hydropower re-operation, especially for large dams such as Lower Sesan 2, should be prioritized53. Alternative energy sources and optimizing hydropower generation of existing dams will be increasingly important as hydrologic intensification expected with climate change reduces hydropower generation from existing dams and reduces the benefits of proposed dams54.An alternative to future dams in the Sekong Basin is to promote other alternative renewable resources like solar energy or to re-operate existing dams to maximize hydropower generation without new dams. For example, solar energy combined with centralized power management and trading could enable Thailand, Laos, and Cambodia to meet future energy demand without new hydropower dams55. Like the LMB, the hydrology of the 3S Basin has well-defined wet (May–October) and dry (November–April) seasons. More than 80% of annual rainfall occurs in the wet season. Across the 3S Basin, mean annual rainfall varies greatly from ~ 1500 mm in the lower reaches to ~ 2500 mm in the southern headwaters and > 3000 mm in the northern headwaters56.The 3S Basin is one of the Mekong’s main tributaries and drains southern Laos, Vietnam’s central highland, and northeastern Cambodia, joining the Mekong mainstem in Stung Treng Province, Cambodia Fig. a. The 3S57.Fish monitoring was conducted from June 2007 to May 2014 in the 3S Basin. Each of the three monitoring sites, that could extend a few kilometers in length, was located in each 3S river adjacent to the village where participating fishers were based  each day (12 ± 2 h per day) were used in this study. Fish were identified and counted using a fish species list (∼ 900 species) from the MRC Mekong Fish Database, FishBase, and other existing literatureIn this study, daily fish samples were averaged across all fishers for monitoring sites to daily mean samples, where were then aggregated into monthly fish community data. Monthly data were used to capture fish biodiversity response to seasonal hydrology and climate. Daily data added noise, but did not change monthly trends. Descriptive statistics including mean, standard deviation, minimum and maximum monthly fish species richness and abundance , and differences between the monitoring sites are provided in Table rda function of the “vegan” R package59. We used Hellinger-transformed data of the total monthly fish community using the decostand function of the “vegan” R package59. This transformation reduced the impact of the highest fish abundance values60. PCA reduces dimensionality and summarizes the main features of a dataset. For example, data points may separate into clusters when samples are taken from different seasons or ecosystems or gradients of sites when a given biotic community is adapted to different conditions of the ecosystems60.To assess spatial-seasonal changes in fish biodiversity in the 3S Basin, a principal component analysis (PCA) was performed using the indicspecies” R package61 to determine the indicator fish species and compute and test their indicator values against a P value of 0.05 based on a permutation test60. If any species has a significant indicator value, it is considered an indicator species. An ecosystem that supports a high number of indicator species can provide information on the characteristics and environmental conditions of the habitats and sites that they share61. A species was considered an indicator when its occurrence and mean abundance in the study site remained high throughout the study period, implying favorable ecological conditions at that site throughout the study period60. In this regard, a river that supports a number of fish indicator species may indicate that the river’s ecological conditions are favorable and stable over time. Otherwise, the fish species occurring in the river are merely visitors or do not occur consistently in the river due to hydrological or water quality fluctuations, habitat alteration, or river fragmentation.We also determined indicator fish species for each 3S river. An indicator fish species here is a species that occurs consistently through time, and more abundant compared to other species. Indicator species value ranges from 0 to 1. Indicator values near 0 indicate rare or sporadic abundance. When a species is consistently abundant in sites and is found at all sites of that group, and therefore has a large mean abundance within the group, its indicator value is 1. Smaller indicator values suggest that a species is not an indicator species; however, smaller indicator values do not necessarily preclude species from being indicators because the value also depends on the abundance of the species caught in each sample. We used the “www.fishbase.se), and threatened fish species are listed as Critically Endangered (CR), Endangered (EN) and Vulnerable (VU)62. The fish biodiversity metrics such as species richness, abundance and diversity (refereeing to Shannon’s diversity index) were used for all three fish categories for each 3S river. Species richness and species abundance refer to the number of different species and the total individuals of all species per sample or per given area, respectively. The Shannon’s diversity index is computed following the equation: diversity = -1Σ [pi × ln (pi)]27. Here, pi is the proportion of individuals of the ith species in a given community, and is computed as pi = n/N, where n is the individuals of the ith species and N is the total number of individuals in the whole community. The metrics were compared to understand whether fish communities in the Sekong, Srepok, and Sesan Basins are distinct from one another. The comparison was based on one-way ANOVA using the aov function in the “stats” R package, or Kruskal–Wallis tests using the kruskal.test functions63. The former was applied when residuals of the Shapiro–Wilk test were normal; otherwise, the non-parametric test was used. When significant differences were detected, we performed Wilcoxon tests using the wilcox.test function of the “stats” R package63 to evaluate the differences in fish biodiversity metrics among river basins.Fish biodiversity was grouped into three fish categories: (1) total species, (2) migratory species and (3) threatened species. Each category differs in sensitivity to river conditions, river connectivity, and imminent risk to biodiversity. Migratory fishes undergo regular, predictable movements from one region to another within a larger system , Cambodia Fisheries Administration, and all methods were carried out in accordance with relevant guidelines and regulations of IFReDI. Fish catch and handling after all scientific measurement and records was granted to local fisher for consumption and sale by Fisheries Capture Committee, and the need for ethics approval is deemed unnecessary according to the regulations of IFReDI.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.


The average length of hospitalization was 18 ± 8 days. Reasons for hospitalization included orthopedic-related issues (28%), spinal cord injury (28%), stroke (20%), and other (23%). In this sample, 23% had prior sleep disorders (mostly sleep apnea), and 60% came from an acute care unit. Patients’ sleep quality was assessed using the Pittsburgh sleep quality index (PSQI) at admission and before discharge. Demographic and medical data were collected. Patients’ mental health status was also assessed at the same intervals. Nighttime sound levels and the average number of sleep disturbances were also collected throughout the study (6 months). Results: Our data revealed that most patients had poor sleep (PSQI > 5) at admission (86%) and discharge (80%). Using a repeated ANOVA, a significant interaction was obtained between sleep quality and the presence of a diagnosed sleep disorder . The sleep quality of patients with sleep disorders improved over their stay, while the sleep of patients without such disorders did not. The mean nighttime sound collection level averages and peaks were 62.3 ± 5.1 dB and 86.1 ± 4.9 dB, respectively, and the average number of sleep disturbances was 2.6 ± 1.1. Conclusion: The improved sleep observed in patients with vs. without sleep disorders might be related to the care received for treating such disorders over the stay. Our findings call for the better detection and management of poor sleep in acute inpatient rehabilitation settings. Furthermore, if our findings are replicated in the future, studies on the implementation of quiet times for medical staff, patients, and family should be performed to improve sleep quality in the inpatient rehabilitation setting.Objectives: In this preliminary, longitudinal study, our objective was to assess changes in sleep quality during an inpatient stay in a rehabilitation setting in the United States and to relate changes to patients’ demographic and clinical characteristics . Methods: A total of 35 patients participated in this preliminary study (age = 61 ± 16 years old, 50% <65; BMI = 30 ± 7 kg/m Sleep is an essential component of health, especially after an initial illness or injury during rehabilitation. Studies have established how the deprivation of sleep affects body-wide metabolism and hormone regulation . FurtherScheduled rest and sleep seem to promote relaxation and reduction of stress levels . HoweverThis prospective, longitudinal study was based on data obtained during assessments using validated scales see  among paData were collected from February 2018 to August 2018. The inclusion criteria were the following: (1) able to complete verbal assessments; (2) 18 years old or more. The exclusion criteria were the following: (1) medical conditions that require intensive nursing care during the nighttime sleep period as determined by the attending physician ; (2) medical proxy unavailable to consent. All patients were admitted to the acute rehabilitation hospital from home, nursing facilities, or other hospitals. This prospective study was approved by the Institutional Review Board of the Casa Colina Hospital and Centers for Healthcare (CCHCH).The Pittsburgh sleep quality index (PSQI) was the primary outcome measure of this study. It is a self-report questionnaire that assesses sleep quality over a time interval. The measure consists of 19 individual items, creating 7 components that produce 1 global score and takes 5–10 min to complete. The PSQI is intended to be a standardized sleep questionnaire for clinicians and researchers to use with ease and is used for multiple populations. Clinical studies have found the PSQI to be reliable and valid in the assessment of sleep disorders ,21.The patient-reported outcomes measurement information system (PROMIS) provides clinicians and researchers access to reliable, valid, and flexible measures of health status that assess physical, mental, and social well-being from the patient perspective. This measure consists of 10 items to produce 1 global score and takes about 2–5 min to complete. PROMIS was established in 2004 with funding from the National Institutes of Health (NIH) as one of the initiatives of the NIH Roadmap for Medical Research .The Beck Anxiety Inventory (BAI) is a 21 multiple-choice self-report inventory that is used for measuring the severity of anxiety symptoms. The questions included in this scale cover common symptoms of anxiety that occurred over the past week. The BAI is designed for individuals who are of 17 years of age or older and takes 5 to 10 min to complete. Each response is scored on a scale value of 0  to 3 (severely). Higher total scores indicate more severe anxiety symptoms .The Patient Health Questionnaire-9 (PHQ-9), a tool specific to depression, scores each of the DSM-5-related criteria based on the mood module from the Primary Care Evaluation of Mental Disorders (PRIME-MD). It includes 9 questions measuring their depressive symptoms with response options ranging from 0  to 3 (nearly every day). Higher scores suggest greater symptomology. The PHQ-9 takes about 5–10 min to complete and is both sensitive and specific in its diagnoses, which has led to its prominence in the primary care setting and in research studies .Nighttime sound levels (dB) and the average number of sleep disturbances within the acute inpatient rehabilitation setting were also obtained over four months. A total of 16 recordings were obtained once every week on the same day around 1 a.m. in the hall and inside the patient rooms using a sound pressure level meter.p < 0.05. Finally, descriptive statistics (average and standard deviation) were used to report nighttime sound levels and the average number of sleep disturbances within the acute inpatient rehabilitation setting.To assess the changes in sleep quality across time, repeated ANOVA measures were applied using PSQI total scores at admission and discharge as outcome variables. Interactions between changes in PSQI total scores and demographic/medical variables  were tested. The association between patients’ mental health status and changes in the total PSQI scores was also assessed using the total scores of the PROMIS, BAI, and PHQ-9 at both admission (pre) and discharge (post). All results were considered significant at mean = 61 ± 16 years old, 51% < 65 years old; BMImean = 30 ± 7 kg/m2; 51% female; 51% Caucasian). The average length of hospitalization was 18 ± 8 days. The reasons for hospitalization included orthopedic-related issue , spinal cord injury , stroke , and other . In this sample, 23% (n = 8) had prior sleep disorders  and 54% (n = 19) came from an acute care unit  obtained a score of > 5 at admission and only 20% of patients (n = 7) obtained a score of <5 at discharge. Using Chi-square as a secondary analysis, the change in proportion of patients with scores of >5 did not change significantly from admission to discharge . Moreover, a significant interaction was observed between changes in total PSQI scores and diagnosed sleep disorders , where the presence of a disorder was associated with an improvement in sleep quality throughout the hospital stay. Post hoc analysis was performed to probe this significant interaction using the Bonferroni–Holm method and resulted in a significant difference between PSQI total scores at the two time points for patients with a diagnosed sleep disorder (pholm = 0.009) but not for patients without a diagnosed sleep disorder (pholm > 0.05).The main effect of the changes in the total PSQI scores at admission versus discharge was not observed [F  = 0.317, p > 0.05). Patients’ demographic variables such as age, gender, BMI, and ethnicity were also not related to changes in total PSQI scores , length of stay , BMI , gender , ethnicity , pre-hospital living setting , or reason for hospitalization  between groups (with versus without a diagnosed sleep disorder).To assess differences in patients’ characteristics between groups (diagnosed sleep disorder versus no diagnosed sleep disorder) at the first time-point, a t-test was performed for continuous variables  and a Chi-square test for dichotomic variables . We did not find any difference in terms of age  to changes in PSQI total scores see .The mean nighttime sound collection level averages and peaks were 62.3 ± 5.1 dB and 86.1 ± 4.9 dB, respectively, and the average number of sleep disturbances was 2.6 ± 1.1.Our objective was to assess changes in sleep quality during an inpatient stay in a rehabilitation setting and to relate changes to patients’ demographic and clinical characteristics. According to our data, the majority of patients had poor sleep (PSQI > 5) at admission (86%) and at discharge (80%). None of the variables considered seemed to significantly impact the sleep quality of patients, except the presence of a sleep disorder. Indeed, based on our results, the sleep quality of patients with sleep disorders seemed to improve over the inpatient stay. These findings are novel, as previous studies have not assessed the sleep quality of patients in the presence of sleep disorders in such a clinical setting ,26.Sleep disorders  are more frequent in patients with spinal cord injury  and stron = 2000) which led to a high effect resolution when compared to this study. In addition, BMI is an established predictor of poor sleep quality [Our preliminary study has several limitations. First, this unicentric study had a relatively small sample size and calls for a multicentric study with a bigger sample to confirm our findings and increase generalizability. A significant difference in the main effect in sleep quality was not found but might have been due to the small number of time-points in this prospective design. Having more time points would allow for a greater detailed view of how sleep quality changes temporally. Future studies could also include measures, such as the functional independence measure (FIM), to relate changes in sleep quality to rehabilitation outcome . Our pre quality ; however quality , althoug quality . Finally quality , levels  quality , or depr quality , which cIn summary, sleep is an essential component of health, and disrupted sleep is among the most common complaints of patients that survive a critical illness, impacting the rehabilitation process. Our preliminary study confirms that patients with a diagnosed sleep disorder should have their sleep quality closely monitored and also suggests that the sleep quality of inpatients admitted in an acute rehabilitation unit should be more systematically assessed and improved (if needed) throughout their stay. Our findings regarding nighttime sound levels and sleep disturbances advocate for the implementation of quiet time in such settings. However, further investigations are warranted. In the future, multicentric studies performed with a larger sample size will allow for better control of demographic, medical, and mental health related variables and should lead to a better understanding and management of the sleep quality in inpatient acute rehabilitation settings.


Vibrio harveyi, even better than that of loaded PB hydrogels. Here, we provide evidence for the potential capacity of Chitosan_ZnO hybrid hydrogels for the preventive and curative treatment of diseases that impact aquaculture animal health and prevent drug resistance by bacteria.In this study, we synthesized and characterized pH-responsive Chitosan–AgCl-doped ZnO hybrid hydrogels and evaluated their potential for loading aquaculture bioactive compounds, and assessed their antimicrobial properties against a threatening pathogen associated with disease across a broad spectrum of warm water fish and invertebrates. Hydrogel characterization consisted of assessing morphology via SEM, composition via EDS, hydrogels’ network components interactions via FT-IR and pH response through swelling behavior determinations. The swelling characterization of the synthesized hydrogels demonstrated a pH-responsive behavior, showing that low pH values caused the hydrogel polymeric network to expand and capture more of the aqueous solution. These characteristics make the synthesized hydrogels suitable for the encapsulation and controlled release of drugs and bioactive compounds in aquaculture. Chitosan_ZnO hybrid hydrogels showed great antimicrobial activity against  Vibrio spp.In recent years, aquaculture has developed worldwide ,2. HowevVibrio harveyi, associated with disease across a broad spectrum of warm water fish and invertebrates.Alternatively, so-called bioactive compounds include both nutrients and non-nutrients present in the food matrix  that can produce physiological effects beyond their classical nutritional properties . In aqua3(CH2)11SH)) solution in methanol ; then, this solution with PE substrates was put in a 60 °C water bath for 15 min. Subsequently, the PE substrates were removed from the dodecanethiol solution and immersed in the colloidal solution of ZnO nps previously prepared for one minute and then removed from the nps solution. The solvent was evaporated from the substrates via direct heating on watch glass on a hot plate at 60 °C for 10 min. Next, we prepared a 3 mM equimolar precursor solution of zinc nitrate hexahydrate 2·6H2O), Sigma Aldrich) and hexamethylenetetramine  in water. Finally, to achieve ZnO doping, silver nitrate at 2 at. % (2 atoms of Ag for every 100 Zn atoms) and 0.1 N HCl (enough to reach a pH of 5.5) were added to the precursor solution under constant stirring. Once the precursor solution was ready, PE substrates with nps were immersed in it. Finally, the precursor solution with the PE substrates was subjected to a microwave treatment for 60 min at 300 W. The samples obtained were rinsed with distilled water and dried at room temperature. The AgCl co-doped ZnO material grown on the PE substrates was collected with a spatula for future experiments.First, we obtained a colloidal solution of ZnO nps as carried out previously . Subsequv/v) of glacial acetic acid  in distilled water was added to the chitosan solution with constant stirring until a homogeneous and viscous solution was obtained. Moreover, 0.2% (wt./v) sodium tripolyphosphate  (TPP) aqueous solution was prepared and pH was adjusted to 3 with HCl 1.0 N. This solution was poured into the viscous chitosan solution until an opalescent coloration was obtained; this hydrogel sample was labeled as chitosan. Using a syringe, drops of the last solution with similar volume and weight (8 ± 0.2 g) were deposited on silicon wafers and allowed to stand for 24 h.First, a solution with 0.2 g of chitosan  in 20 mL of distilled water was prepared. After, 2%  using a JEOL-JIB 4500 microscope . Prior to SEM observation, the hydrogel drop samples on silicon wafers were coated with an Au/Pd thin layer in a vacuum evaporation chamber. The elemental analysis was carried out in situ using an energy dispersive X-ray (EDS) microanalysis  at an electron accelerating voltage of 15 kV.−1. For this, 20 uL of hydrogels samples were put on a KBr plate and sandwiched under another KBr plate to spread the liquid thin layer between the plates. The spectrum of a clean KBr plate (without hydrogel sample) was used for subtraction (background spectrum).FT-IR spectra of liquid hydrogel samples were recorded in a Bruker Tensor 27 Spectrometer , in a wave number range from 4000 to 400 cm0 is the initial hydrogel weight. The statistical analysis to evaluate the loading capacity of the hydrogels consisted of a design relying on two factors (hydrogel composition and pH) and was applied with 2 and 5 levels, respectively, in which the dependent variable, the swelling percentage, was evaluated in triplicate. IBM SPSS Statistics 27.0.0 versión software was used to carry out the analysis and consisted of a two-way ANOVA with α = 0.05, where the assumptions of normality and homoscedasticity were corroborated by the Shapiro–Wilk and Levene tests, respectively. To verify the statistical differences between the pH levels, Tukey’s multiple comparison test was used with a significance of 0.05.To evaluate how much solution could be loaded into the synthesized hydrogels, a series of 5 aqueous solutions with adjusted pH values of 2, 4, 6, 7, and 8 (with NaOH and HCl) was prepared. Subsequently, an amount of each synthesized hydrogel was weighed in triplicate and each portion was placed in one of the previously prepared aqueous solutions with a different pH. Then, each hydrogel was removed from the aqueous solutions and weighed (excess water on the surface of the hydrogel was previously removed with wipes) every 10 min for 30 min. Finally, the swelling percentage was determined using the following expression:v/v). New hydrogels, now with PB loads, were prepared as previously described. Each PB aqueous solution together with the ZnO solution was poured into the chitosan solution right before the acetic acid solution. Finally, this produced Cx- and Lc PB-loaded hydrogel samples, which were labeled as Chitosan_ZnO_Cx and Chitosan_ZnO_Lc, respectively.The phytobiotic (PB) products used were from Alivira-Karizoo Laboratories , Licorol (Lc), whose main components were menthol (89 g/mL) and eucalyptol (92.2 g/mL); the other product used was Coxsan (Cx), composed mainly of carvacrol, thymol, and allicin. Aqueous solutions of each PB products were prepared in a 1:1000  was used to assign the highest percentage of similarity among type strains.To evaluate the antimicrobial properties of the synthesized hydrogels, the minimum inhibitory concentration (MIC) and its antimicrobial activity against Vibrio harveyi strain (1 × 108 CFU/mL). A 10 µL aliquot of each hydrogel was placed (in triplicate) and incubated at 35 °C for 24 h to observe growth. For each treatment, the inhibition halo diameters were measured, in millimeters.To determine the antimicrobial activity of the hydrogels, a plate with Mueller–Hinton solid medium was inoculated with the Vibrio harveyi (1 × 108 CFU/mL) was added into each tube and then incubated at 35 °C for 24 h. After incubation, the tubes were visually examined to identify the first tube with turbidity. Then, 100 µL was taken and inoculated into plates with Muller–Hinton medium from the three tubes prior to the tube in which turbidity was identified. The plates were incubated at 35 °C for 24 h. After incubating, the number of CFU (Colony Forming Units) from each plate were counted in order to determine the MIC of each hydrogel. Growth media, bacterial culture, and antibiotic were used as a positive control, while growth media and bacterial culture were used as the negative control.To determine the lowest concentration of hydrogels that produced an absence of growth in plates after 24 h of incubation, the standard microbiological tube dilution method was performed. Serial dilutions of the hydrogels were made (with concentrations ranging from 200 to 0.39 µg/mL of each hydrogel) in Eppendorf tubes with 200 µL of Mueller Hinton liquid culture medium (MH). Once the hydrogel dilutions were prepared, 200 µL of The morphology of the AgCl-doped ZnO samples was expected as follows; arrangements of pin shape clusters formed of rod structures with diameters from 300 nm to 1.0 µm a,b. In tRegarding the chemical composition analysis of the synthesized hydrogels, Zn, O, C, and some other elements derived from their synthesis were detected in the EDS analysis . Discard−1 region, attributed to the O-H and N-H groups stretching vibrations [−1 correspond to stretching vibrations of aliphatic groups (-CH2 and -CH3) of the chitosan pyranose ring [−1 which corresponds to C=N (nitrile) stretching vibrations [−1 [−1 were attributed to O-P-O asymmetric stretching of TPP phosphate groups [−1 [−1 bands are related to C-O bonds in cyclic compounds like glucose [−1 an absorption band corresponding to asymmetric Zn–O vibration appeared [−1, 1564 cm−1 and 1407 cm−1 are attributed to the amide I (C=O stretching), amide II (N-H bending) and amide III (C-N stretching) bonds vibrations, respectively [The chemical interactions between the components of the synthesized hydrogels were analyzed via FT-IR . The Chibrations . The Chiose ring . A weak brations . TPP phoions [−1 . Additioe groups . Both hyoups [−1 . The 107 glucose ,55. Centappeared . The banectively ,59,60,61ectively ,58. The ectively  but alsop < 0.05) for both hydrogel compositions, highlighting that both the Chitosan and Chitosan_ZnO hydrogel had better swelling capacity in acidic solutions. Multiple comparison tests following the Tukey method found that the swelling percentage at pH values of 6, 7 and 8 were not statistically different (p > 0.05). The polymeric network of the chitosan hydrogel using TPP as a crosslinking agent can be formed due to the physical crosslinking through hydrogen bonding and dipole–dipole interactions between neighboring ester groups and chitosan chains [+H3) of the chitosan hydrogel causes the polymeric network to expand, which allows the hydrogel to capture more water [The swelling behavior of hydrogels provided an overview of the strength and capacity of the polymeric network formed by the components of the hydrogel. The hydrogen proton concentration of the medium in which the hydrogel is located can determine the ionization of various functional groups that make up the polymeric network, affecting the hydrogel’s properties. According to the swelling behavior results , a signin chains . Howeverre water . This isre water ,66, loadre water . Figure Vibrio harveyi is shown in The lowest concentration of hydrogels at which there was no visible growth of Vibrio harveyi. This shows that all synthesized hydrogels had inhibitory effects against these bacteria and that Chitosan_ZnO hydrogels had a powerful inhibitory effect by themselves even without PB loading. They also reveal that Chitosan_ZnO hydrogels loaded with Cx PB presented a small decrease in their antimicrobial activity. These results were not expected considering the antimicrobial properties of the PBs and the Chitosan_ZnO hybrid hydrogels. It should be noted that the pH of the culture medium was close to 7 and according to the swelling behavior of the synthesized hydrogels, in these conditions, the hydrogels allowed for the release of the encapsulated compound. However, the lower antimicrobial activity in the PB-loaded hydrogels may be due to the proton release by the chemical components of PBs, which could cause a pH decrease in the culture medium. As already determined for the synthesized hydrogels, low pH values favor the interaction between the components of the hydrogel, creating a stronger network that does not allow the PB to escape easily, inhibiting it from contributing to the antimicrobial activity of the hydrogel. Furthermore, it is also possible that due to this acidity, the hydrogel with Cx PB had less antimicrobial activity than the hydrogel with Lc. This could be caused by the hydroxyl group of phenol present in the compounds carvacrol and thymol (main components of Cx), which is more acidic than the hydroxyl group of cyclohexanol in menthol (a component of Lc) [t of Lc) .Vibrio harveyi, even better than that of loaded PB hydrogels. Protons releasing from PB in loaded hydrogels probably cause a stronger encapsulation, inhibiting the release of the PBs. However, here, we provide evidence for the potential capacity of Chitosan_ZnO hybrid hydrogels for the preventive and curative treatment of diseases that impact aquaculture animal health and prevent drug resistance by bacteria. Further research is needed on the direct application and effect of hydrogel capsule administration on commercially important aquaculture organisms.In this work, pH-responsive Chitosan AgCl co-doped ZnO hydrogels for the encapsulation of drugs and bioactive compounds were prepared. The synthesized porous polymeric network of Chitosan_ZnO hybrid hydrogels is formed via possible interactions between chitosan functional groups, TPP phosphoric ions and ZnO NRs that form a bigger network with more interactions and a better swelling behavior. The response of the hybrid hydrogel to a different pH solutions was evaluated through swelling behavior determinations and showed that low pH values cause the hydrogel polymeric network to expand and capture more of the aqueous solution. Meanwhile, neutral and basic pH values cause the hydrogel to shrink. These characteristics make the synthesized hydrogels suitable not only for the encapsulation and controlled release of drugs and bioactive compounds in aquaculture but also for other animals and humans. Chitosan_ZnO hybrid hydrogels showed great antimicrobial activity against


TM Air is a second-generation laryngeal mask whose shaft angle can be adjusted after insertion. Since the device’s performance has been assessed on mannequins only, this study aimed to evaluate SingularityTM Air’s effectiveness in the clinical setting. The prospective single-center cohort study included 100 adults undergoing elective surgery under general anesthesia and suitable for airway securing with a laryngeal mask. The primary endpoint was the oropharyngeal leak pressure, and the secondary endpoints were the ease of insertion and the patient’s comfort. Laryngeal mask insertion was successful in 97%, and mechanical ventilation was possible in 96% of patients. After insertion, the median (IQR) oropharyngeal leak pressure was 25 (18–25) cm H2O, which remained stable at 25 (25–25) cm H2O after 20 min. The median (IQR) time for successful manual bag ventilation was 42 (34–50) seconds. Nineteen patients complained of side effects , but none persisted. The SingularityTM Air performed well in a clinical setting, and its oropharyngeal leak pressure was comparable to that of other masks reported in the literature. The time for successful manual ventilation was slightly longer, and patients reported more temporary side effects.A laryngeal mask is one of the most widely used airway management devices. The Singularity Since its introduction in the early 1980s, the laryngeal mask has quickly became one of the most widely used airway management devices in anesthesia ,3. ImproMany studies have been published comparing different laryngeal masks, including systematic reviews and meta-analyses ,6. In moThe Singularity™ Air laryngeal mask  and registered with German Clinical Trials Register (DRKS00029113). The study was conducted from January to June 2023 at a Swiss acute care hospital where approximately 9000 general anesthetics are performed annually.2 based on institutional standards) or symptomatic esophageal reflux disease. According to institutional guidelines, surgery in which the patient is not supine or is in the Trendelenburg position, laparoscopies, and ear–nose–throat surgery are unsuitable for airway securing by laryngeal masks. Further exclusion criteria were pregnancy, emergency surgery, loose teeth, inability to follow the investigation procedures , participation in another study, or previous enrolment in the current investigation. All patients provided written informed consent to participate in the study.Patients undergoing elective surgery under general anesthesia and determined to be suitable candidates for a laryngeal mask based on the judgment of the attending anesthesiologist were assessed for inclusion. The patient-based inclusion criteria were: American Society of Anesthesiologists (ASA) physical status I–III, fasting according to internal guidelines , and ≥18 years of age. The primary exclusion criterion was any contraindication to using a laryngeal mask, such as morbid obesity , and slightly bending the shaft.The anesthesia induction was performed according to institutional guidelines. Patients were given 3.75 to 7.5 mg midazolam orally 30 min before being transferred to the operating room, where they received standard monitoring ), a peripheral venous cannula for the administration of fluids and medications, and connection to an EEG-based Bispectral Index (BIS) anesthetic monitor . 2) of 0.8 with the patient breathing independently. The anesthetic was administered as a propofol-based, total intravenous anesthesia (TIVA) with a target-controlled infusion (TCI) syringe pump based on the Schnider pharmacokinetic model  that uses the patient’s age, weight, height, and gender. The standard setting for the anesthesia induction was a target effect-site propofol concentration (Cet) of 6 mcg/mL and infusion of remifentanil 0.1 mcg/kg/min. For the induction of anesthesia, 1–3 mcg/kg of fentanyl was administered intravenously. Before placement of the laryngeal mask, a BIS value of lower than 60 and the possibility of gentle bag–mask ventilation were required. After securing the airway, the propofol Cet was reduced to maintain a BIS value of between 40–60 while factoring in the patient’s blood pressure, heart rate, and general clinical observations. Before starting the anesthetic, patients were preoxygenated by tightly applying a ventilation mask to their face and administering oxygen up to an end-tidal fractionated oxygen concentration . If manual ventilation by bag was insufficient after insertion of the laryngeal mask, the remedial steps were as follows: induce deeper anesthesia, reposition the laryngeal mask, increase the bend in the tube, and use neuromuscular blocking drugs. If insertion or manual ventilation after insertion were still not possible, another airway device was used .The exact time point for insertion of the laryngeal mask was determined by the responsible anesthesiologist. The laryngeal mask was inserted, as suggested by the manufacturer, with the patient’s head slightly extended. A second person assisted in opening the patient’s mouth by performing the Esmarch maneuver. If insertion was not possible on the first attempt, the following steps were taken: induce deeper anesthesia, change insertion technique , or use neuromuscular blocking drugs. After insertion of the laryngeal mask, its cuff was filled with air up to a cuff pressure of 60 cm H2, positive end-expiratory pressure (PEEP) 5 cm H2O, inspiratory oxygen concentration (FiO2) 0.6) according to institutional standards. If mechanical ventilation was deemed insufficient by the responsible anesthetist due to inadequate tidal volume or unreliable CO2-tracking, measures were taken to adapt ventilator parameters , and manipulate the laryngeal mask. A gastric tube , lubricated with sterile gel , was advanced through the gastric lumen of the Singularity™ Air. Successful gastric placement of the probe was verified by auscultation over the stomach when insufflating air using a 50 mL syringe. At the end of surgery, emergence from general anesthesia and removal of the laryngeal mask were performed according to institutional guidelines.The patient was then mechanically ventilated  of the circle circuit was then set to 40 cm H2) to less than 90% was recorded. At the end of the anesthetic, the laryngeal mask was removed, and the tip was inspected for the presence of blood. The duration of the anesthetic was documented. We recorded the time from stopping bag–mask ventilation in order to start airway manipulation to the first successful manual ventilation by bag through the laryngeal mask. Successful ventilation was defined as thoracic–abdominal movement followed by an appropriate capnography signal. Any drop of the peripheral oxygen saturation  to rate throat soreness on a scale from 0  to 10 (worst sore throat imaginable). In addition, we asked patients if they were experiencing difficulties when swallowing or dysesthesia in the mouth or pharynx, and if they required treatment to alleviate symptoms. Patients were advised to contact the study team should any discomfort persist.p-value of <0.05 was considered statistically significant. All analyses were conducted in Stata .Data are presented descriptively as mean and standard deviation or median and interquartile range based on normality tests (Shapiro–Wilk test). Comparisons between time points were performed using the Wilcoxon signed-rank test. A Forty-one of the 100 patients included in the study were fitted with a size 4 laryngeal mask and the remaining 59 were fitted with a size 5. The patients’ ages ranged from 18 to 88 years. Study participants underwent the following surgical procedures: 31% orthopedic, 25% urologic, 22% visceral surgery, 20% gynecological, and 2% mixed procedures. 2O  and size 5 = 24 ). The oropharyngeal leak pressure approximately 20 min after the first measurement improved for the entire cohort (p = 0.0003)  to 25  cm H2O. These same values were observed for sizes 4 and 5, which also showed improvements .At the initial assessment after laryngeal mask insertion, the median oropharyngeal leak pressure was 25  cm Hn = 15), bending the tube (n = 5), providing deeper sedation (n = 5) or neuromuscular relaxation (n = 1), making a second attempt (n = 5), or repositioning the patient’s head (n = 1). Insertion of the laryngeal mask was not successful in three patients ; therefore, an alternative airway device had to be used.No difficulties occurred during insertion in 65% of the patients . For most of the remaining patients, insertion was successful after altering the insertion technique, such as guiding the laryngeal mask by finger or inserting a catheter in the gastric lumen , the tube had to be bent (n = 3), the laryngeal mask had to be repositioned (n = 1), or deeper sedation was needed (n = 1). The median time required for successful manual bag ventilation through the laryngeal mask was 42 (IQR 34–50) seconds (44 (IQR 39–59) in size 4 and 40 (IQR 32–48) in size 5 laryngeal masks). Oxygen saturation did not drop below 90% for any patients before bag ventilation through the laryngeal mask was established.Manual bag ventilation through the laryngeal mask was successful in 92% of the patients. For five additional patients (n = 3) or applying Esmarch maneuver (n = 1)) was needed to insert the tube. In six patients, it was not possible to insert a gastric tube (4 (10%) with laryngeal mask size 4 and two (5%) with size 5). Mechanical ventilation succeeded in 78 of the 97 patients (80%). In an additional 18 patients (20%), bending the laryngeal mask’s tube or modifying the respirator settings was required to facilitate mechanical ventilation. Only one patient needed an alternative airway device. The median duration of anesthesia was 70 (IQR 58–93) minutes. When removing the laryngeal mask, the tip was stained with blood in 23% of the cases . On the first day after surgery, seven patients complained of a sore throat that they rated above three on a ten-point verbal analog scale (2 (5%) with size 4 and 5 (8%) with size 5 laryngeal masks). Nine patients reported difficulties swallowing after anesthesia (4 (10%) with size 4 and 5 (8%) with size 5 laryngeal masks). Pharyngeal dysesthesia after anesthesia was reported by three patients (2 (5%) with size 4 and 1 (2%) with a size 5 laryngeal mask). None of these complications required therapy, and none of the patients reported back because of persistent discomfort after hospital discharge or after the telephone call on postoperative day one.2O, was well established at the time of insertion and its effectiveness further improved slightly 20 min after insertion.Laryngeal masks are important tools for airway management in anesthesia and emergency medicine ,3. This 2O as our maximum pressure. This is a clinically meaningful threshold, given that peak inspiratory pressure is often set to a maximum of 20 cm H2O for mechanical ventilation. Even though the leak per se would not be a reason to change a laryngeal mask, a prolonged leak of a certain amount would eventually result in unreliable respiratory monitoring, unreliable tidal volumes, and (if administered) leakage of inhalational anesthetic agents. Our study cohort had a similar median leak pressure of 25 cm H2O after insertion and after 20 min. However, the distribution shifted upwards and the range of values narrowed, resulting in statistically significant improvements in the leak pressure. It is possible that the warming of the laryngeal mask causes some material softening, which results in a better adjustment to the pharyngeal structures. These outcomes were comparable to other studies reporting similar values [2O in our study, the leaking pressure may have been affected [Given the high insertion and ventilation success rates of laryngeal masks in general, leak pressure is a more meaningful parameter to measure ,7,9. Comr values ,15,16,17affected . AlthougWe report a relatively long time-to-adequate ventilation compared to other studies. In some studies, this time was only 4 s when using another laryngeal mask , which is not possible in some second-generation laryngeal masks . A gastric tube with a larger diameter may be advantageous when gastric content other than air needs to be removed.After removal, blood at the tip of the laryngeal mask is a sign of injury, caused mainly by the insertion of the airway device. While blood on the tip is a commonly reported outcome, it has not been directly linked or correlated with patient discomfort. Our rate was within the range of other published studies, but our patient population reported higher values for side effects such as a sore throat, pain when swallowing, and hoarseness. It is worth noting that our patients did not receive neuromuscular blockers. Fortunately, none of these complaints required therapy or persisted. As previously mentioned, the relative stiffness of the tube may have contributed to these outcomes.Our study had some limitations that ought to be mentioned. First, there was no strict protocol followed for managing cases of insufficient leak pressure. As a result, the effectiveness of bending the shaft of the laryngeal mask Singularity™ Air under these conditions remains unclear. The same applies to the effect of bending the shaft during gastric tube insertion or for tracheal intubation through the device. Second, while a study population of 100 is within the range of other studies comparing devices and evaluating leaking pressures, it may be inadequate for detecting safety issues. Third, although this study was designed as a descriptive clinical evaluation of a new device, using a comparison group would have been advantageous and should be the next step for further studies. Lastly, the size of the laryngeal masks was selected depending on the patients’ weight. This is what the manufacturer suggests, but in reality, it may be advantageous to select the size depending on height and/or gender.In conclusion, the recently introduced laryngeal mask Singularity™ Air performed relatively well in a clinical setting, but may not have outperformed the earlier generation or other second-generation devices. Since we did not compare the device to other laryngeal masks, further studies may be warranted. Specifically, investigations should focus on (1) closely assessing the functioning of the unique rotary wheel , its ability to effectively bend the device after insertion, and its impact on improving airway sealing; (2) evaluating the possibility of tracheal intubation through the Singularity™ Air; and (3) selecting the appropriate size of the device.