Amynthas triastriatus (Oligochaete: Megascolecidae) is a widely distributed endemic species in Southern China. To shed light on the population genetic diversity and to elucidate the population differentiation and dispersal of A. triastriatus, a population genetic structure study was undertaken based on samples from 35 locations collected from 2010 to 2016. Two exclusive lineages within A. triastriatus—lineage A and lineage B—were revealed. Lineage A was mainly distributed at high altitudes while lineage B was mainly distributed at low altitudes in Southeast China. The genetic diversity indices indicated that the populations of A. triastriatus had a strong genetic structure and distinct dispersal histories underlying the haplogroups observed in this study. Combined with morphological differences, these results indicated a new cryptic subspecies of A. triastriatus. Lineage A was almost degenerated to parthenogenesis and lineage B had a trend to parthenogenesis, which suggested that parthenogenesis could be an internal factor that influenced the differentiation and dispersal of A. triastriatus. The divergence time estimates showed that A. triastriatus originated around Guangxi and Guangdong provinces and generated into two main lineages 2.97 Ma (95%: 2.17–3.15 Ma) at the time of Quaternary glaciation (2.58 Ma), which suggested that the Quaternary glaciation may have been one of main factors that promoted the colonization of A. triastriatus. Understanding the evolutionary history of a species is a fundamental goal of evolutionary biology  and manyEarthworms are very important macro-invertebrate detritivores that tend to remain in the same areas during long periods of time and show low ability to cross mountains; for these reasons they are often an ideal model for phylogeographical study ,5,6,7,8.Their structural simplicity makes it difficult to distinguish earthworm species accurately using only morphological characteristics . MoleculAmynthas longisiphonus, Amynthas corticis, Amynthas gracilis and Metaphire californica) revealed that mitochondrial ATP8 exhibited the highest evolutionary rate and the NADH gene was second to it [In recent research, mitochondrial COI, COII, NDI, 12S rRNA and 16S rRNA have been widely used to study the phylogenetic evaluation, mitochondrial diversity and population genetic diversity of earthworm using multivariate concatenation ,27,28,29nd to it . Nuclearnd to it ,32. The nd to it ,17,24,33Amynthas triastriatus (Chen 1946) has two pairs of spermathecal pores in segments 7/8–8/9 and belongs to the aeruginosus-group. It is named after the triangle formed by three genital papillae in each male pore zone and is a widely distributed endemic species in Southern China [A. triastriatus that are similar in morphology due to a tendency to parthenogenesis; for example, some populations have a thin and lustreless seminal chamber and prostate glands absent, and others have a plump and glossy seminal chamber and small prostate glands. As a result, their classification status and the genetic relationships are obscure.rn China . There aA. triastriatus originated in the vicinity of Guangxi and Guangdong provinces then split into two lineages around 4.31 Ma (3.24–5.37 Ma) [A. triastriatus presents two lineages, one displays parthenogenesis and the other is degenerate towards parthenogenesis. This raised general questions as whether there are cryptic species, whether the population genetic structure are useful to clear the classification status of A. triastriatus and whether genetic isolation occurred between the two lineages due to population expansion and reduction along with ancient geographical events. In the present study, a fragment of mitochondrial gene COI of 65 individuals collected from 35 locations and one Aporrectodea species, and 4691bp segments comprising mitochondrial genes COI, COII, ATP8, ND6, 12S rRNA, 16S rRNA and NDI from 35 locations and one Aporrectodea species were used to determine: (1) the population genetic diversity and the classification status of A. triastriatus; (2) the population differentiation and dispersal of A. triastriatus; (3) the time of divergence and colonization histories of A. triastriatus, as well the main factors impacting these.A previous study based on only seven individuals indicated that the ancestor of 5.37 Ma) —this wasA. triastriatus were collected by digging at 35 different locations  was removed and cleaned carefully under a stereomicroscope for DNA extraction, and the rest of the individual was used for taxonomic identification.A total of 232 individuals of ocations , Table 1® Mollusc DNA kit  according to the manufacturer’s instructions. The quality of DNA samples was checked by electrophoresis using a 1% agarose gel and stored at −20 °C.Total genomic DNA was extracted using the E.Z.N.A.® HIFI DNA Polymerase , 4 μL dNTP, 5 μL HiFi Buffer, 2 μL primer and 35.4 μL ddH2O. The PCR of genes COI, COII, ATP8, 12S rRNA, 16S rRNA and NDI were carried out for 5 min at 94 °C followed by 32 cycles of 94 °C for 30 s, 50 °C for 30 s and 72 °C for 1 min, with an extension of 10 min at 72 °C. The PCR of gene ND6 was carried out for 5 min at 94 °C followed by 40 cycles of 94 °C for 30 s, 51.9 °C for 30 s and 72 °C for 1 min, with an extension of 10 min at 72 °C. The primers of mitochondrial COI, COII, 12S rRNA, 16S rRNA and NDI are listed in The mitochondrial genes were amplified using 50 μL of reaction mixture with 1 μL template DNA, 0.6 μL Trans TaqThe PCR products were visualized on a 1% agarose gel and subsequently purified then sequenced by the Beijing Genomics Institute  using an ABI 3730 DNA analyzer. The sequences were compared with known earthworm sequences in GenBank using the BLAST search algorithm ,36,37,38Aporrectodea species as an outgroup. Time of divergence and colonization history were conducted using a combination of COI, COII, ATP8, ND6, 12S rRNA, 16S rRNA and NDI datasets with one Aporrectodea species as an outgroup. Sequences of COI and a combined dataset were aligned using the Clustal X 1.8 program [Population genetic structure and genetic diversity studies were conducted using single-gene datasets of COI with one  program .Models for sequence evolution and corresponding parameters were estimated using jModeltest v.2.1.7 . The besA. triastriatus individuals and one individual of Aporrectodea trapezoides as outgroup. ML analyses were performed using PhyML v.3.0 [The phylogenetic trees were reconstructed using different methods based on single-gene COI datasets of 65 ML v.3.0 ; clade sML v.3.0 ; the parPopulation structure was evaluated in Arlequin v.3.5.2  via AMOVD [Fs [Basic statistics of mtDNA diversity, including nucleotide and haplotype diversity, Tajima’s D  and Fu’sD [Fs  neutraliD [Fs . BayesiaD [Fs  and TracD [Fs  to inferA. triastriatus were estimated using BEAST 1.8.2 [Aporrectodea trapezoides as an outgroup. A previous study has estimated 2.1 My−1 as the average substitution rate for COI, COII, ATP8, ND6, NDI, 12S and 16S genes [A. triastriatus and Aporrectodea trapezoides was set at a mean age of 212 Ma (95%: 200–230 Ma); this represented the minimum age of Lumbricoidea and Megascolecoidea in our tree and is considered to be when the breakup and drift of Gondwana occurred [The Bayesian tree and the time of divergence of ST 1.8.2  based on6S genes , and thioccurred ,51,52,53occurred . The treA. triastriatus fell into two exclusive lineages, A and B, and this result was strongly supported in both the BI tree (1.00) and ML tree (100%). Lineage A consisted of four clades (clades 1–4) and lineage B consisted of two clades . Clade 1 was mainly distributed in Guizhou, Hubei, Chongqing and Sichuan provinces , clades 2 and 3 were mainly distributed in Anhui and Fujian provinces  and clade 4 was mainly distributed in Guangxi, Guangdong and Hunan provinces . Clade 5 was mainly distributed in East China, such as Zhejiang, Anhui province, east of Jiangxi and Fujian province  and clade 6 was mainly distributed in the west of Southern China, such as Sichuan, Hunan, Guangdong, Guangxi and Guizhou provinces .The posterior probabilities from the Bayesian analysis and the bootstrap values from the ML analysis were plotted on the ML tree . A. triaA. triastriatus had two exclusive lineages, which was in concordance with the phylogenetic trees . Amynthas triastriatus became two main lineages about 2.97 Ma (95%: 2.17–3.15 Ma) (As shown in 3.15 Ma) , Table 4A. triastriatus was studied using AMOVA to check structure at the lineage level based on a fragment of the COI gene , indicating significant population genetic structure. The value of pairwise comparisons of ΦST among lineages was significantly high, suggesting a great genetic differentiation exists between lineages A and B.Hierarchical population structure in COI gene . The perD values were negative within lineages, but positive and significant between lineages, indicating that the COI gene was possibly not a neutral evolution. Fu’s Fs test is more sensitive to detect population expansion [Fs test values were negative and nonsignificant within lineages, indicating that lineages A and B had experienced demographic expansion. Fu’s Fs test values were positive and nonsignificant between lineages, indicating that A. triastriatus had experienced demographic expansion. The results of nucleotide mismatch distribution analysis based on fragments of COI, COII, ATP8, ND6, 12S rRNA, 16S rRNA and NDI genes showed the following: a transition between unimodal and bimodal distributions for COI and 12S, the same as expected for COII, 16S, ND1, ATP8 and ND6 of lineage A; unimodal distribution for COI and ND1, the same as expected for COII, 16S, ATP8 and ND6; and bimodal distribution for the 12S gene of lineage B. The results revealed that lineage A , named Amynthas triastriatus usualis subsp. nov.; the description for this proposed subspecies is included as the final part of this paper.Taxonomically speaking, COI gene divergence of ea longa ,59. The ea longa . In thisParthenogenesis is a unisexual reproduction pattern corresponding to bisexual reproduction that is present in a few animal and plant species. Generally, the loss of sexual reproduction has been thought a dead end in evolution, leading to early extinction ,61. But A. triastriatus, lineage A with a thin and lustreless seminal chamber and no prostate gland observed was almost degenerated to parthenogenesis, while lineage B with a plump and glossy seminal chamber and small prostate glands had a tendency to parthenogenetic reproduction. Compared to their sexual relatives, parthenogenetics often occur at high latitudes, high altitudes, on islands or island-like habitats, in xeric environments or in disturbed habitats [ST values indicated that lineages A and B had a high degree of isolation and a significant genetic differentiation exists with lineage A having greater genetic diversity than lineage B. These findings supported those of Simon et al. (2003), that unisexual lineages with high genetic diversity can live in wider geographical ranges than their sexual ancestor.Parthenogenesis often results in polymorphism in earthworms , with mohabitats ,73,74,75habitats . For exaA. triastriatus located at the terminal node of the terminal clade of the fourteenth group [Based on phylogenetic studies, parthenogenetic lineages usually occupy terminal nodes of phylogenetic trees; therefore, unisexual forms are usually assumed to arise from sexual congeners . The phyth group . In the th group ,53,77.Aporrectodea trapezoides (95%: 200.41–229.89 Ma) was not significantly different from previous estimates of the diversification between Lumbricidae and Megascolecidae at about 200–220 Ma [pheretima spread into China from the Indo-China Peninsula, then spread to the east or north [A. triastriatus and GX201201-11A  was estimated to have taken place 6.49 Ma (95%: 5.23–7.88 Ma) in Guangxi province [The divergence time estimate of 215.28 Ma for outgroup 0–220 Ma ,51,52,53province .A. triastriatus then formed into two main lineages (A and B) at around 2.97 Ma (95%: 2.17-3.15 Ma), which happened together with the Quaternary glaciation (2.58 Ma). The diversification of lineage A was divided into three stages.It is proposed that Liquidambar species during the Quaternary glaciation [A. triastriatus, significantly several refuge should exist during this period. Therefore, the Heishiding Mountains is proposed as a potential refugium for lineage A.The first stage  occurred during MIS 7, 8, 9 and 10 during the Penultimate Glacial Period and before MIS 6, during which glaciation remained widespread with the maximum temperature decrease experienced in Western China and alpine areas ,82,83,84o biloba ,86, and The third stage occurred during the last glacial period (MIS 2-4), in which the Gonghe Movement (~0.15 Ma BP) occurred between the Middle and Late Pleistocene to raise the Tibetan Plateau to its present height, High-Asia experienced several temperature plunges, and glaciation existed in most of the mountains in western China ,88,89,90Unlike lineage A, lineage B mainly dispersed to the east. The diversification of lineage B was also divided into three stages. The first stage  occurred during MIS 6 and 8, two glacial periods of the Penultimate Glaciation, with lineage B still dispersed in Zhejiang province. The third stage, as for lineage A, occurred during the last glacial period (MIS 2-4). One route of lineage B dispersal was to Anhui and Jiangxi provinces and another route was north to Guizhou and Guangxi provinces. Geological and climatic changes during this period had a profound effect on the colonization of lineage B, and it is also possible that the distant dispersal of B12 was affected by other factors such as birds or human beings.Drawida japonica Michaelsen, 1892 were analysed using 79 samples obtained from the Shandong and Liaodong peninsulas. Results show that the intraspecific genetic diversity of D. japonica has been influenced by geographic isolation [Hormogaster elisae Álvarez 1977) collected from the central Iberian Peninsula and some invasive species , Amynthas gracilis ) collected from a volcanic island show that environmental factors may have some influence on earthworms’ genetic evolution [A. triastriatus formed about 0.01 Ma, with its dispersal process totally covered by the Quaternary glaciation period. This period had a major impact on climate and vegetation in Southeast Asia through the interaction of temperature, rainfall and topography [A. triastriatus. Drawida japonica, H. elisae, A. corticis, A. gracilis and A. triastriatus are wildspread species, ancient geography event, climate changes, geographic isolation  and environment factors should play important role in the differentiation and diffusion of wildspread earthworm species.The population structure of solation . The resvolution ,94. The volution ,96,97. Avolution ,99. The pography ,101. We Amynthas was carried out using 77 Amynthas species from South China. It has been determined that at least one branch of Amynthas spread to the southeast of China, and another spread to the southwest of China [A. triastriatus was consistent with the research of Sun et al., with lineage A widely distributed in the southwest of China, and lineage B widely distributed in the southeast of China. Overall, the colonization routes of A. triastriatus were from south to north, which is in accord with the known dispersal direction of earthworms in China [The phylogenetic evaluation of the genus of China . In the in China ,102.Amynthas triastriatus (Oligochaete: Megascolecidae) of the aeruginosus-group is a widely distributed endemic earthworm species in Southern China. Population genetic structure exhibited a very high degree of isolation and two exclusive lineages based on existing specimen and gene data. Lineage A was almost degenerated to parthenogenesis, lineage B had a tendency to parthenogenesis. There were distinguish genetic differentiations exist between lineages and lineage A had high haplotype diversity and strong genetic structure, additionally, lineage A mainly distributied at high alitude areas. These results indicated a new cryptic species of A. triastriatus; combined with morphological differences, a new subspecies of A. triastriatus is proposed. And the Quaternary glaciation may be one of main factors that promoted the colonization of A. triastriatus. In future, there may be more studies of differentiation and dispersal of A. triastriatus including more populations based on this study, and the population genetic structure analysis should play an important role in the earthworm taxonomy.Amynthas triastriatus it is proposed that a new subspecies be established that encompasses the populations of lineage B.Based on the above results, to unravel the classification puzzle of Material. Holotype: 1 clitellate (C-FJ201111-04A): China, Fujian Province, Meihua Mountain Nature Reserve , 1207 m asl, black soil beside road, 13th July, 2011, J. B. Jiang, J. Sun, X. D. Lei and H. W. Feng coll. Paratypes: 85 clitellates: 2 clitellates (C-FJ201111-04B) same date as for holotype. 4 clitellates (C-FJ201004-01): China, Guangxi Province, Youxi Country , 952 m asl, black soil beside road, 24th Aug, 2010, J. B. Jiang coll. 1 clitellate (C-GD201115-04): China, Guangdong Province, Lianping county , 410 m asl, brown soil under eucalyptus trees, 04th Aug, 2011, J. B. Jiang coll. 12 clitellates (C-GX201319-06): China, Guangxi Province, Jiuwan Mountain Nature Reserve , 1226 m asl, yellow soil, 18th May, 2013, J. P. Qiu, Y, Hong, J. B. Jiang, L. L. Zhang and Y. Dong coll. 4 clitellates (C-GZ201609-02): China, Guizhou Province, Fanjing Mountain National Reserve , 1316 m asl, yellow soil under bryophyte beside road, 21th May, 2013, J. P. Qiu, J. B. Jiang, L. L. Zhang and Y. Dong coll. 2 clitellates (C-HU201309-02): China, Hunan Province, Bamian Mountain Nature Reserve , 1034 m asl, black soil under willow trees, 21th Jun, 2013, J. P. Qiu, J. B. Jiang, L. L. Zhang and Y. Dong coll. 2 clitellates (C-JX201302-04): China, Jiangxi Province, Longhu mountain , 185 m asl, black soil beside road, 15th Jun, 2013, J. B. Jiang, L. L. Zhang, Y. Dong and S. Lei coll. 19 clitellates (C-JX201311-01): China, Jiangxi Province, Jinggang Mountain National Reserve , 1300 m asl, black soil under bamboo forest beside road, 17th Jun, 2013, J. B. Jiang, L. L. Zhang, Y. Dong and S. Lei coll. 4 clitellates (C-SC201004-06): China, Sichuan Province, Gongga Mountain National Reserve , 1558 m asl, brown soil beside river, 06th Aug, 2010, J. P. Qiu, J. B. Jiang coll. 1 clitellate (C-ZJ201101-02): China, Zhejiang Province, Tianmu Mountain National Reserve , 1052 m asl, black soil beside road, 02nd Nov, 2011, J. B. Jiang, J. Sun, L. L. Zhang coll. 1 clitellate (C-ZJ201511-01): China, Zhejiang Province, Jinhua City , 160 m asl, black soil beside road, 08th July, 2015, Y. Dong, M. S. Chen, X. Gao and C. Peng coll. 4 clitellates (C-ZJ201515-03): China, Zhejiang Province, Yunhe City , 290 m asl, yellow soil beside road, 09th July, 2015, Y. Dong, M. S. Chen, X. Gao and C. Peng coll. 1 clitellate (C-ZJ201517-02): China, Zhejiang Province, Wenzhou City , 710 m asl, brown soil beside road, 09th July, 2015, Y. Dong, M. S. Chen, X. Gao and C. Peng coll. 6 clitellates (C-ZJ201518-01): China, Zhejiang Province, Yandang mountain , 120 m asl, brown soil beside road, 10th July, 2015, Y. Dong, M. S. Chen, X. Gao and C. Peng coll. 10 clitellates (C-AH201601-01): China, Anhui Province, Huangshan City , 325 m asl, brown soil, 08th May, 2016, J. B. Jiang, J. Sun, Y. Dong and Y. Zheng coll. 2 clitellates (C-AH201609-02): China, Anhui Province, Huangshan City , 120 m asl, yellow soil, 09th May, 2016, J. B. Jiang, J. Sun, Y. Dong and Y. Zheng coll. 10 clitellates (C-AH201610-01): China, Anhui Province, Huangshan City , 146 m asl, brown soil, 09th May, 2016, J. B. Jiang, J. Sun, Y. Dong and Y. Zheng coll.Diagnosis. Dimensions 120–150 mm by 4.9–6.8 mm at clitellum, segments 108–111. First dorsal pore in 11/12. Setae numbering 20–26 at III, 24–30 at V, 30–34 at VIII, 40–48 at XX and 55–60 at XXV; 12–16 between male pores; seta numbering between spermathecal pores 10–14 at VIII. Two pairs of spermathecal pores in 7/8–8/9, 1/3 circumference ventrally apart from each other. One pair of male pores in XVIII, 1/3 circumference apart ventrally, each on the top of a raised, round porophore surrounded by 3–5 circular ridges. Two presetal and two postsetal collapse-topped genital papillae present on XVIII. Ampulla oval-shaped, stout duct as long as 1/5 ampulla. Diverticulum as long as 1/3 main pouch, terminal 1/2 dilated into an oval-shaped glossy seminal chamber. Prostate glands small or only one side observed.Description. External characters: Brown pigment on dorsum, no pigment on ventrum. Dimensions 125 mm by 5.1 mm at clitellum, segments 111. Prostomium 1/2 epilobous. First dorsal pore in 11/12. Setae numbering 22 at III, 26 at V, 32 at VIII, 44 at XX and 58 at XXV; 14 between male pores; 14 at VIII between spermathecal pores. Setal formula: AA = 1.2 − 1.4AB, ZZ = 1.6 − 2.0ZY. Clitellum annular, taupe on dorsal, in XIV–XVI, setae not visible external. Two pairs of spermathecal pores in 7/8–8/9, 1/3 circumference apart ventrally. Two pairs of postsetal genital papillae present on VII, two pairs presetal and two pairs postsetal on VIII, three pairs presetal on IX, 1/3 circumference ventrally apart from each other . One pair of male pores in XVIII, 1/3 circumference apart ventrally, each on the top of a raised, round porophore surrounded by 3–5 circular ridges, two presetal and two postsetal genital papillae on median side. Two big collapse-topped genital papillae present on XVIII, 1/4 circumference ventrally apart from each other. (h other. A. SingleInternal characters. Septa 5/6–7/8, thick and muscular, 10/11–12/13 slightly thickened, 8/9–9/10 absent. Gizzard bucket-shaped, in IX–X. Intestine enlarged distinctly from XV. One pair of intestinal caeca in XXVII, brown, simple, dorsal margin smooth, 17 short pointed saccules in ventral margin, extending anteriorly to XXIII. Four pairs of esophageal hearts in X–XIII. Ovaries in XIII. Two pairs of spermathecae in VIII–IX, about 1.7–2.0 mm long. Ampulla oval-shaped, stout duct as long as 1/5 ampulla. Diverticulum as long as 1/3 main pouch, terminal 1/2 dilated into an oval-shaped glossy seminal chamber. (chamber. B. Holandchamber. C.Ethymology. The species is named after the existence of prostate glands.Remarks. There are certain differences between the new subspecies and A. triastriatus. For instance, the new subspecies has small prostate glands that are absent in A. triastriatus, and the seminal chamber of the new subspecies is plump and glossy while thin and lustreless in A. triastriatus. Additionally, the first dorsal pore of the new subspecies is located at 11/12, but at 10/11 in A. triastriatus. Furthermore, the new subspecies has more papillae in both the spermathecal and male pore regions than A. triastriatus, and more finger sacs in the ventral intestinal caeca than A. triastriatus.


Familial hypercholesterolemia (FH) is a genetic disorder of cholesterol metabolism that affects an estimated 1/250 persons in the United States and abroad. FH is hallmarked by high low-density lipoprotein (LDL) cholesterol and an increased risk of premature atherosclerotic cardiovascular disease. This review summarizes recent global evidence showing the utility of FH genetic testing across diverse populations. Clinical and other qualitative outcomes following FH genetic testing were improved FH diagnosis, treatment initiation or continued treatment, treatment modification, improved total or LDL cholesterol levels, education on lifestyle management, and genetic counseling. This summary of evidence should be considered by those seeking overall evidence and knowledge gaps on the utility of FH genetic testing from a global perspective and for certain ethnic and age populations. These findings can be used to inform insurance policies and coverage decisions for FH genetic testing, policy recommendations to reduce the clinical and public health burden of FH, clinical practice and guidelines to improve the management of FH populations, and ongoing research involving FH genetic testing. We conclude that further investigations are needed to examine: (1) non-clinical outcomes following FH genetic testing; (2) patient-reported outcomes following FH genetic testing to convey patient experiences, values, and goals; and (3) clinical outcomes following FH genetic testing in non-Caucasian and pediatric populations in the United States and abroad. Familial hypercholesterolemia (FH) is a genetic disorder of cholesterol metabolism that is associated with very high cholesterol levels  > 200 mg/dL or 5.2 mmoL/L; LDL cholesterol > 130 mg/dL or 3.4 mmoL/L) from birth ,7. VariaAn individual with FH may experience various symptoms depending on the associated genotype. Heterozygous individuals can display typical cardiovascular disease symptoms, which include arterial plaques  and premature atherosclerotic cardiovascular disease  . Homozygκ = 0.993 and 0.958, respectively) and reported high sensitivity (91.9% and 100%) and high specificity (100% and 99.9%) [An International Classification of Diseases, Tenth Revision, ICD-10) code for FH diagnosis was recently approved in October 2016 . Diagnos0 code fod 99.9%) . Reportsd 99.9%) . Among tFH genetic testing is recommended because the results have the potential to change or influence patient management and identify at-risk first-degree, biological relatives . In 2018n = 1). Titles and abstracts were screened by one investigator (RHS). Full original research article reports were searched in PubMed/MEDLINE, EMBASE, and Yale University’s TRIP Medical Database, and Google Scholar in February of 2020 using Boolean string with MeSH terms, Diagnosis AND “Genetic Testing” AND “Hyperlipoproteinemia Type II.” Given that evidence on the utility of FH genetic testing spiked after 2016, based on our observations of “results by year” from a PubMed search, filters were used to find studies published after 2016 to ensure that the findings were not only relevant but timely. We carefully selected and reviewed studies reporting clinical and non-clinical outcomes following FH genetic testing in humans. Studies that did not report at least one clinical or non-clinical outcome were excluded. Original research articles were excluded if they were not published in the English language and if they were case reports , number of participants screened, study participants’ age groups , geographic location, study participants’ ethnicities, FH diagnosis criteria used, clinical diagnosis, and reported phenotypes. We also extracted clinical and non-clinical outcomes, including patient reported outcomes (PROs) that followed genetic testing. Lastly, we determined, among studies meeting our inclusion criteria, if cascade screening was used to recruit family members for FH genetic testing, following the identification of probands/index cases. A total of 352 articles were retrieved from a search in PubMed/MEDLINE. After screening against the exclusion criteria, nine articles were selected for review. A search in EMBASE returned 519 results; however, after applying our inclusion/exclusion criteria and removing duplicates, 10 articles were selected for review. A TRIP Medical Database search returned zero results after screening against the inclusion/exclusion criteria. Two articles were found through a search in Google Scholar. A total of 21 articles were reviewed. n = 56,332 or 92.5% of total clinically suspected cases). The most represented continents include North America (mainly a US population in Pennsylvania), Europe, and Asia, although actual population ethnicities were rarely reported. DLCN was the most common clinical diagnostic criteria used (14 studies). FH phenotypes reported were LDL cholesterol > 130 mg/dL (or 3.4 mmoL/L), total blood cholesterol > 200 mg/dL (or 5.2 mmoL/L), xanthoma, xanthelasma, corneal arcus, and overt CAD . Five clinical outcomes following FH genetic testing were described or reported: improved FH diagnosis, treatment initiation or continued treatment, treatment modification, and improved total or LDL cholesterol levels. Only eight studies reported patient education on lifestyle management and genetic counseling following FH genetic testing, which we considered as non-clinical outcomes.Across these 21 studies, FH genetic testing was performed among a total of 60,893 clinically suspected adult and pediatric individuals across 11 countries in North and South America (83.40%), Europe (14.58%), Asia (1.55%), and Australia (0.45%). Studies largely reflect individuals tested in Italy, the US (Pennsylvania only), and Estonia (91.3% combined), and predominantly Caucasian populations. Clinically suspected cases presented across all 21 studies consisted of largely adult-only populations  and were: (1) concerns about receiving genetic test results and (2) out-of-pocket costs associated with FH genetic testing. PRO reporting was not consistent across these two studies; Pang et al. explored reasons why some parents in their study declined genetic testing for their children, citing parent concerns about possible stigmatization of genetic testing, while Jones et al. conducted qualitative interviews with seven patients to understand the patients’ overall experiences in receiving FH genetic test results ,28. Cascade screening was performed in 12 of the 21 studies, which occurred in Italy, Spain, Slovak Republic, India, Estonia, China, Vietnam, and Western Australia ,37,38,39FH genetic testing is not a new concept to FH diagnosis; however, its utility to address therapeutic challenges concerning FH have been unclear. The utility of genetic testing in any context is greater when genetic test results are actionable in that testing can inform better treatment decisions along with clinician and patient decision-making . Here weOur findings are consistent with a recent systematic review and meta-analysis showing that FH genetic testing can confirm FH diagnosis over clinical criteria alone, depending on the diagnostic algorithm and the method of analysis . Howeverp < 0.0001), placing the Black population at a possibly higher risk of premature cardiovascular disease due to undiagnosed FH [Lipid levels in individuals can vary based on several factors , thus rendering clinical diagnostic criteria unsuitable in some populations of FH variant carriers . In factnosed FH . TherefoWe also examined age characteristics across all 21 studies (adult versus pediatric (≤18 years), or both) and found that the study populations consisted of mostly adult populations . This finding shows that pediatric populations are largely absent from such investigations. Therefore, future research should examine clinical and non-clinical outcomes following FH genetic testing in children globally. Cascade screening is a robust, cyclical, and family tracing and screening mechanism that is used to identify an index/proband case and family members at risk for a genetic condition like FH . In sligRace and geographic ethnicity/ancestry can provide clues about a person’s or population’s lifestyle, culture, and exposome, making race and/or ethnicity possible determinants of health outcomes seen among populations that share a specific genotype, phenotype, and/or culture. Studies presented in our review largely reflect cases in Italy, the US (Pennsylvania only), and Estonia (91.3% combined), and Caucasian populations. It is unlikely, however, that these cases reflect the general global FH population. Also, given that the two studies conducted within the US in the state of Pennsylvania occurred within the same health system, it is possible that the population reflected in Jones et al.’s study overlaps with the population reflected in Abul-Husn et al.’s study. Therefore, we recommend that future studies ascertain such outcomes in more geographically, ethnically, and health system diverse populations, especially since geography and ethnicity can be predictors of age of FH diagnosis, which might affect clinical outcomes and management .Understanding PROs following FH genetic testing is important, as expert groups including the American College of Cardiology recommend that FH genetic testing should be offered to individuals of any age in whom a strong clinical index of suspicion for FH exists based on the examination of the patient’s clinical and/or family histories . PROs caClinical guidelines for FH genetic testing have been established by the National Lipid Association (NLA) in 2011, the International FH Foundation in 2014, and the American Association of Clinical Endocrinologists and American College of Endocrinology (AACE/ACE) in 2017 . These gStudies in other global regions not covered in this review are underway or beginning to examine the clinical and actionable outcomes discussed herein, to ascertain the clinical and public health utility of FH genetic testing. For example, the gulf familial hypercholesterolemia registry study is occurring among an adult population within the Arabian Gulf region where the prevalence of probable and definite FH (per DLCN criteria) is 1/232 . Such stn = 462, mean age of 51 years) that a TG level of 4 mmol/L showed a negative predictive value for FH at 100% (p < 0.0001) [On the other hand, some studies have sought to determine when FH genetic testing is unnecessary. For example, as part of a national plan to make FH genetic testing available to all lipid clinics in England, English researchers sought to determine a triglyceride (TG) level at which FH is unlikely . They de 0.0001) . The res 0.0001) . Researc 0.0001) . In addi 0.0001) . There are limitations to our literature search and review. First, the literature reviewed was searched and identified by a single reviewer (RHS) without comparison with a second reviewer. Therefore, the evidence summarized should be confirmed via discussion with the authors of the articles presented or by reviewing the articles directly. Further, the research articles presented herein were not reviewed for methodological quality before we summarized the evidence of clinical utility. Nonetheless, our review provides an important summary of the latest evidence on the utility of FH genetic testing. This review can be useful alongside other literature examining barriers to FH genetic testing and can be used to guide global calls to action, such as the Global Call to Action on Familial Hypercholesterolemia, and public policy recommendations to reduce the clinical and public health burden of FH ,59. Clinicians and insurers should consider this summary of evidence when determining (1) knowledge gaps on the patient-reported outcomes following FH genetic testing, evidenced by an unclear picture or inconsistent reporting of non-clinical outcomes following FH genetic testing; (2) if FH genetic testing may be appropriate in clinically suspected cases and how PROs are or can be used to understand what is needed to reach desired treatment outcomes; and (3) the clinical utility of FH genetic testing from a global perspective and across certain ethnic and age populations. Forthcoming clinical guidelines should also be based on the latest evidence herein. Finally, more research is needed to examine and determine the role of non-clinical outcomes and PROs following FH genetic testing, identify resources needed to inform treatment goals and help improve treatment outcomes in FH patients, and assess clinical outcomes following FH genetic testing in non-Caucasian and pediatric populations in the US and abroad.


Oral microbiota composition and functions of non-enzymatically lysed oral bacteria samples were evaluated using 16S rRNA gene sequencing and functional bioinformatics tools in the adolescents. Adaptive immune responses were assessed by testing for serum IgG antibodies against a panel of common oral bacteria in adults. In the adolescents, host genetic factors were associated with both the detection and abundance of microbial species, but with considerable variation between species. Host genetic factors were associated with predicted microbiota functions, including several functions related to bacterial sucrose, fructose, and carbohydrate metabolism. In adults, genetic factors were associated with serum antibodies against oral bacteria. In conclusion, host genetic factors affect the composition of the oral microbiota at a species level, and host-governed adaptive immune responses, and also affect the concerted functions of the oral microbiota as a whole. This may help explain why some people are genetically predisposed to the major dental diseases of caries and periodontitis.Maintaining a symbiotic oral microbiota is essential for oral and dental health, and host genetic factors may affect the composition or function of the oral microbiota through a range of possible mechanisms, including immune pathways. The study included 836 Swedish twins divided into separate groups of adolescents ( Bacterial communities in the oral cavity are among the most dense and diverse in the human body ,2. StudiThe main colonization of the gastro-intestinal canal takes place after birth and stabilizes into niche-specific polymicrobial biofilms during childhood . SeveralPrevotella pallens, Corynebacterium durum, and species in genera, including the Veillonella, Pasteurellaceae, and Abiotrophia genera  bacterium HMT 349. Among species with a statistically significant additive genetic contribution for both abundance and detection were Streptococcus mutans  and Scardovia wiggsiae , which are found to associate with caries in various populations, including Sweden [Stomatobaculum longum , which is found to be associated with caries development in S. mutans carrying-adolescent in Sweden [S. mutans, strong ICC scores (0.99) were seen in MZ caries-affected young twins but not in MZ caries-free or DZ twins  were found for g Sweden ,40, and n Sweden  (Tables DZ twins e.n = 3258 KO functions), the ICC values were higher in MZ twins than DZ-SS twins, indicating that genetic factors were associated with predicted function (p < 0.0001). The pattern was similar for the MZ twins compared to DZ-OS (p < 0.0001). The top 100 functions based on ICC-values  were evaluated for protein-protein interaction networks (PPI) and functional enrichment using the String database. PPI analysis indicated an overall functional enrichment (p = 2.4 × 10−12) with enhancement in the KEGG pathways linked to carbohydrate metabolism, e.g., fructose and mannose metabolism (p = 7.4 × 10−7), glucose transport system (PTS) (p = 1.2 × 10−5), and starch and sucrose metabolism (p = 0.0077) (We used PICRUSt  to gener 0.0077) b.p = 5.4 × 10−10) for carbohydrate metabolism and functions associated with fructose and mannose metabolism (p = 1.2 × 10−7), the glucose transport system (PTS) (p = 2.8 × 10−5), and starch and sucrose metabolism (p = 0.033). A list of ICC and ACE model estimates for the 100 evaluated functions is found in Next, heritability was estimated by ACE modeling of the top 100 predicted functions c. AdditiAggregatibacter actinomycetemcomitans and Haemophilus parainfluenzae (p = 0.031), with the most marked difference for the two Porphyromonas gingivalis and the Fusobacterium periodonticum strains  and the F. periodonticum strain (0.76), explaining over 75% of the variance. Component A also explained approximately half of the variance for Actinomyces odontolyticus, Corynebacterium durum, Corynebacterium matruchotii, and Streptococcus oralis  was statistically significant for 47% (nine of the 19 antibody responses), and among these 67% (six of the nine antibody responses), more than half of the variance could be attributed to A . The adds oralis a.Streptococcus mutans, Scardovia wiggsiae, and Stomatobaculum longum . AdditiIn the present study, we aimed to evaluate the impact of host genetic factors on the oral microbiota and the immune response to oral bacteria with the intention to deepen the understanding of the driving forces behind oral biofilm formation and the potential consequences of the oral microbiota. Twin-based studies are an established methodology for heritability research questions that use naturally occurring genetic variations to estimate the relative importance of genetic and environmental factors. For one arm of this study, we chose young twins who were reared together and were still living at home at the time of participation in the study, while for the other arm, we chose twins in later adult life who may have lived apart for many years and were unrelated to the younger twins. The rationale for applying a split-sample design was that for the microbiota characterization, a situation with “native” microbiota was considered desirable and for this, the young twins in the CATSS cohort in the STR offered a group which was likely to have normal saliva flow, healthy mucosal surfaces, and tooth surfaces that were not significantly disrupted by dental restorations, and where saliva had been collected. For the analyses of immune responses, a study group with serum available and with more extended exposure to both caries and periodontal disease conditions were sought. Here the middle-aged twins in the SALT cohort represented an age group where signs of periodontal disease, and hence colonization of periodontal pathogens were likely. Despite this difference in the design and despite using different outcome measures, we observed some similarities in both arms of the study, in particular, that the shared environmental effects were small and that the major differences in concordance were seen between MZ and DZ twins regardless of whether the twins were of the same or opposite sex.Several studies have examined the gut microbiota in twins and a few targeted the oral microbiota ,41,42,43S. mutans was stable in the mouth over the years, but the numbers varied significantly over time [In line with the gut microbiota , we founver time . Thus, aver time ,48,49.S. mutans, S. wiggsiae and other highly acidogenic species but also might reflect genetic effects on a range of more abundant but less acidogenic species. Interestingly, host genetic associations were stronger for several of these species in MZ twins with caries than without. Though not substantiated in this study, this suggests an interaction between the host genetic effect on S. mutans and the presence of caries. Possible explanations may include the increased penetrance of genetic effects on S. mutans in the presence of environmental features that predispose to caries (gene × environment interaction) or increased penetrance in the presence of genetic risk factors for caries (gene × gene interaction). Notably, heritability for caries is approximately 50% in Swedish twins [S. mutans colonization, they both end up in the caries-free group, and an effect of genotype is not seen.The study found that host genetic factors are strongly associated with predicted carbohydrate metabolism in the oral microbiota. This finding is in agreement with a previous study where the heritability of microbial functions proxied by microbial acid formation was estimated at 76% . Here, tP. gingivalis and also weaker genetic effects on serum antibodies against A. actinomycetemcomitans and F. alocis. These species are all associated with periodontal diseases and have been hypothesized to play a role in general inflammatory conditions, possibly by triggering autoimmunity, such as in rheumatic arthritis (RA) [P. gingivalis has been suggested in RA development by some [A. actinomycetemcomitans produces a pore-forming leukotoxin A that triggers the dysregulated activation of citrullinating enzymes in neutrophils and hypercitrullination [Similar to caries, periodontitis is a disease where oral bacteria and host genetic susceptibility both play a pivotal role. Screening of antibody profiles to 19 oral species in serum from adult twins identified a comparably large heritability estimate for the serum levels of antibodies against two strains of tis (RA) ,51,52,53 by some , due to  by some ,56,57. Slination . The preStreptococcus mitis/Streptococcus oralis/Streptococcus HMT061), and also reported some of the results at the genus level too.The present study has limitations that need to be acknowledged and considered in the interpretation of the results. In line with all twin based studies, the results do not identify specific genes or genomic regions which regulate the oral microbiota, so they do not provide any information about mechanisms. To address this, genome-wide analysis on oral microbiota taxonomy and function would be needed, similar to those which have been undertaken for the gut microbiota . HoweverFinally, a potential cross-reactivity between taxa should be kept in mind when evaluating the checkerboard results. This has been illustrated in a recent publication using the same checkerboard method and set up of bacteria species. That study showed that, among the selected species, the strongest cross-reactivity was among strains of the same species and less between species. Still, it cannot be excluded that shared immune epitopes or non-specific responses could potentially lead to an under- or overestimation of the heritability of the adaptive immune response.The study also holds some evident strengths, such as its relatively large groups of twins with genetically defined zygosity, and the possibility to evaluate equally sized groups of same and opposite-sex dizygotic twins, the combination of deep Illumina sequencing and taxa classification by a curated database that especially targets oral and upper airway species with taxa resolution to the species level for many species. We believe that the split sample strategy is a strength of the study, as it combined adolescents with normal saliva flow, healthy mucosal surfaces, and mostly non-manipulated tooth surfaces, which may represent a “native” microbiota. In contrast, the older adults are in an age group, which is susceptible to periodontitis and more likely exposed to periodontal pathogens which would be beneficial for antibody screening. In the future, we hope to expand the study of adults by relating the antibody responses to periodontal status and screening a wider panel of bacteria.Several aspects of the present study are interesting from a clinical perspective. There were strong host genetic effects on several taxa and metabolic functions relevant to caries in the younger age group, and strong host genetic effects on serum antibody levels to known periodontal pathogens in the older age group. In health, the bacterial biofilms are in equilibrium, but the disruption of the balance into a dysbiotic state may lead to permanent loss of tooth tissues  or tooth-supporting tissues . Thus, tooth biofilm architecture is crucial for oral health or disease, but there is also increasing recognition that oral microbiota may be related to systemic diseases ,61,62. US. mutans and many also Streptococcus sobrinus, whereas in contrasting Swedish adolescents many were S. mutans-free and the prevalence of S. sobrinus was very low [S. mutans in Sweden may not confer the same protection in other settings, and this could be tested through formal interaction analysis. The present results also suggest that genetic factors affect antibody response to a known periodontal pathogen suggested to associate with several general diseased conditions [As anticipated from previous research, the results supported a role of non-shared environmental factors on microbiota composition and function, which is important as lifestyle counseling is a major intervention for dental diseases. While we did not explicitly test for gene-environment interactions in this study, results from sensitivity analysis in caries-concordant groups and results of previous studies suggest that some host genetic factors are more or less important in different situations. Thus, a recent study in a population with high sugar intake, no oral hygiene, rampant caries, and no dental care, all were found to carry very low . Therefonditions , and it nditions .Streptococcus mutans, Scardovia wiggsiae and Stomatobaculum longum  [P. gingivalis was associated with additive genetic influences. These findings provide insight into the genetic and molecular basis of oral microbiota development and the stability, and may help understand the consequences of these for oral and general diseases.The present study supported previous studies in that unshared environmental factors are associated with oral microbiota ecology, but it also identified numerous species where additive genetic influences were strongly associated with being colonized with a bacterial species and the abundance. Species where the detection and abundance were strongly influenced by host genetic factors included caries-associated species, such as escents) . At a mi


In McGill-R-Thy1-APP rats compared to controls, levels of 17β-HSD10–cypD complexes were decreased and those of total amyloid β increased. Moreover, the levels of 17β-HSD10–cypD complexes were decreased in cerebrospinal fluid of individuals with AD (in mild cognitive impairment as well as dementia stages) or with Frontotemporal lobar degeneration (FTLD) compared to cognitively normal controls . Our results demonstrate the weakened ability of 17β-HSD10 to regulate cypD in the mitochondrial matrix probably via direct effects of amyloid β. Levels of 17β-HSD10–cypD complexes in cerebrospinal fluid seem to be the very sensitive indicator of mitochondrial dysfunction observed in neurodegeneration but unfortunately not specific to AD pathology. We do not recommend it as the new biomarker of AD.The nucleus-encoded 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10) regulates cyclophilin D (cypD) in the mitochondrial matrix. CypD regulates opening of mitochondrial permeability transition pores. Both mechanisms may be affected by amyloid β peptides accumulated in mitochondria in Alzheimer's disease (AD). In order to clarify changes occurring in brain mitochondria, we evaluated interactions of both mitochondrial proteins in vitro (by surface plasmon resonance biosensor) and detected levels of various complexes of 17β-HSD10 formed in vivo (by sandwich ELISA) in brain mitochondria isolated from the transgenic animal model of AD (homozygous McGill-R-Thy1-APP rats) and in cerebrospinal fluid samples of AD patients. By surface plasmon resonance biosensor, we observed the interaction of 17β-HSD10 and cypD in a direct real-time manner and determined, for the first time, the kinetic parameters of the interaction ( The nucleus-encoded mitochondrial matrix protein 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10) is an essential protein operating via multiple enzymatic as well as non-enzymatic functions. Its deficiency, marked overexpression or loss of enzymatic function is associated with various pathologies . CytosolD about 40–80 nM and the binding results in a distortion of 17β-HSD10 molecule including deformation of D loop and binding pocket for co-factor nicotinamide adenine dinucleotide ethanesulfonic acid , phosphate buffer , NaCl, KCl, MgCl2, ethanolamine (EA) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich, USA, in molecular biology grade or higher. Ethanol for spectroscopy (purity 99.9% or greater) was purchased from Merck, USA. Oligo-ethylene glycol (OEG) thiols terminated with carboxyl group (HS-C11-(EG)6-OCH2-COOH) and hydroxyl group (HS-C11-(EG)4-OH) were purchased from Prochimia, Poland. N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) were purchased from GE Healthcare, USA. Human recombinant 17β-HSD10 and cypD proteins and monoclonal mouse antibody against mitochondrial cypD (anti-cypD) were purchased from Fitzgerald, USA. Aβ 142 fragment was obtained from AnaSpec, USA. All buffers were prepared using deionized water , phosphate buffer with high ionic strength (PBSNaCl) was prepared from PBS by increasing concentration of NaCl to 750 mM, HEPESBSA was prepared from HEPES by addition of 250 µg/ml BSA, 15 mM KCl and 0.1 mM MgCl2.Sodium acetate  with dispersionless microfluidics , 24 deve2 . SPR chi11-(EG)6-OCH2-COOH and HS-C11-(EG)4-OH thiols was dissolved in ethanol at a total concentration of 0.2 M. SPR chip was immersed in the thiol mixture for 10 min at 40 °C and stored for at least 12 h at room temperature. Then, the chip was rinsed with ethanol and Q-water and mounted into the SPR biosensor. The mixture of 12.5 mM NHS and 62.5 mM EDC (in Q-water) was injected for 10 min to the surface of SPR chip in order to activate carboxylic groups. Then, anti-cypD at a concentration of 5 µg/ml in SA10 was injected for 12 min to covalently attach anti-cypD to the activated carboxylic groups. The surface was consequently washed by PBSNaCl (5 min) to remove the non-covalently bound molecules from a surface of SPR chip and by 0.5 M EA (5 min) to deactivate the remaining active esters.The surface of SPR chips was functionalized with a self-assembled monolayer (SAM) of mixed carboxy-terminated and hydroxy-terminated OEG thiols, on which anti-cypD was immobilized using the amino-coupling as described previously . BrieflyNaCl (5 min) were injected into both channels, followed by HEPESBSA until the stable baseline was obtained. Solutions of 17β-HSD10 at concentrations of 30, 50, 150, 300, and 500 nM in HEPESBSA were prepared and incubated for 10 min at 37 °C to ensure the full tetramerization of 17β-HSD10 [BSA was added to the solution to obtain the 17β-HSD10 at final concentrations of 6, 10, 30, 60, and 100 nM. These solutions were injected for 10 min into both detection (with attached cypD) and reference (without attached cypD) channels to monitor the association phase, followed by exposing the sensor surface to HEPESBSA for 15 min to characterize the dissociation phase. The reference compensated curves (sensor responses from the reference channels were subtracted from those obtained in the particular detection channels) were globally fitted using BIAevaluation software version 4.1 and 1:1 Langmuir model considering mass transport effects. In order to provide the positive control for our biosensor experiments, we performed a control experiment in which we observed the binding of Aβ 1−42 to the cypD immobilized on the SPR sensor surface. In the immobilization step, 2 µg/ml cypD was injected into the detection channel until the sensor response leveled off (~ 5 nm), while MES was continuously pumped through the reference channel. Then, MES (20 min) and PBSNaCl (5 min) were injected into both channels, followed by HEPESBSA until the stable baseline was obtained. In the detection step, Aβ 1−42  was injected into detection and reference channels and the binding was observed for 10 min.Interaction between cypD and 17β-HSD10 proteins was studied in the direct fashion using the detection and reference channels. Initially, MES was injected for 10 min into both channels functionalized with anti-cypD. Then, 2 µg/ml cypD was injected into the detection channel until the sensor response of 1.5 nm was achieved, while MES was continuously pumped through the reference channel. Then, MES (20 min) and PBS7β-HSD10 . Then, 4g for 5 min), supernatants collected and the pellets were washed 3 times with PBS by centrifugation. Samples were then dissolved in Laemli denaturating sample buffer, electrophoresed in precast gradient 5–15% polyacrylamide Mini-Protean TGX gels and blotted to nitrocellulose in Trans-Blot Turbo according to Bio-Rad protocols. Blotted membranes were quenched by incubation in 10% soya milk in PBS, washed, incubated overnight at 4 °C in sealed pouch with either anti-17β-HSD10 or anti-cypD (dilution 1:1000 in both cases), washed and incubated with respective anti-immunoglobulin (IgG)-horseradish peroxidase (HRP) conjugate  for 2 h. To reveal immunoprecipitated proteins, blots were incubated in 20 ml PBS containing 10 mg of 3,3-diaminobenzidine (Sigma) plus 10 µl of 30% hydrogen peroxide and stained membranes were after washing imaged by ChemiDoc XRS + Imager (Bio-Rad).Mixtures of human full length recombinant 17β-HSD10 protein (His tag)  and of human full length cypD protein (His tag)  in PBS  were incubated for 2 h at room temperature and then mixed with 2 µl with either polyclonal rabbit anti-17β-HSD10 (Flarebio) or monoclonal mouse anti-cypD . Incubation was continued for another 2 h and mixtures were then transferred to tubes containing Protein A-sepharose (Sigma) pellet (approx. 50 µl) and incubated while rotating overnight at 4 °C. Samples were centrifuged  and adjusted to mitochondrial protein concentration of 9 mg/ml. Particular aliquots of mitochondria (250 µl) were stored at −40 °C until assayed.Ten 11-month old Tg homozygous male McGill-R-Thy1-APP rats and ten age-matched WT male Wistar controls  or 7-month old male Wistar rats  were housed in cages (2 rats per cage) in a temperature-controlled room (21–22 °C), with a 12:12 h light/dark regime (lights on at 7:00 a.m.) with free access to food (ST-1 diet) and water. All manipulations were performed according to the Guidelines of the European Union Council (86/609/EU). Rats were sacrificed by cervical dislocation, decapitated, and the brains rapidly removed from the skulls. The left hemispheres  were dissected on an ice-cold plate, weighed and immediately used for isolation of mitochondria. Mitochondria were isolated by discontinuous Percoll density gradient . ConcentAll experiments were conducted in accordance with The Declaration of Helsinki. The study was approved by the Ethical Committees of Motol University Hospital, in accordance with the Laws 129/2003 and 130/2003 of the Czech Republic. Written informed consent was obtained from all study participants, either personally or by proxy. CSF samples were collected from 171 patients undergoing lumbar puncture as a part of their routine diagnostic work-up . The solution was applied to 96-well polystyrene plates  and incubated for 4 h at room temperature . Plates were subsequently washed three times with PBS . Nonspecific binding was suppressed by incubation with blocking solution  for 1 h at room temperature followed by overnight incubation at 10 ºC. Prior to the assay, the plates were washed three times with washing solution (PBS and 0.05% Tween 20). A standard curve was prepared using 138 to 4839 ng of rat recombinant protein 17β-HSD10 dissolved in 10% dimethyl sulfoxide. Various volumes of standards, 50 µl of particular samples  and 50 µl of internal standards (prepared in accordance with the samples) were pipetted in duplicates and adjusted to 100 µl by 10% dimethyl sulfoxide. Plates were subsequently incubated with primary antibody  for 4 h at room temperature. Plates were washed four times with washing solution (PBS with 0.05% Tween 20), and secondary antibody  was added. After a 1-h incubation at room temperature, the plates were washed four times with washing solution. Subsequently, substrate solution  was added and plates were incubated for 20 min at room temperature. The reaction was stopped with 0.5 M H2SO4 and plates were measured at the wavelength of 450 nm using an automated plate reader . After the subtraction of the blank, levels of 17β-HSD10 were estimated in particular samples. The detection limit was determined to be about 20 ng of the protein. Intra- and inter-assay variability was determined to be 3.7% and 9.6% at the control protein level of 277 ng.A full length rat recombinant protein 17β-HSD10 (Flarebio) was dissolved in carbonate buffer  were incubated in quadruplicates and measured in accordance with manufacturer’s instructions.Levels of rat cypD were estimated by a kit (MyBioSource). 50 µl of samples  were incubated in duplicates for 5 h. Subsequently, second primary antibody  and lately secondary antibody  were applied. After the subtraction of the blank, the values were compared to those from the internal standards and expressed in percentage.Plate was coated by goat anti-mouse IgG  and then by monoclonal mouse anti-Aβ 13–28 recognizing mouse, rat and human Aβ . 100 µl of samples or internal standards  were incubated in duplicates for 5 h. Subsequently, second primary antibody against cypD  and lately secondary antibody  were applied on the first plate. Analogously, second primary antibody against total Aβ  and lately secondary antibody  were added to the second plate. After the subtraction of the blank, the values were compared to those from the internal standards and expressed in percentage.Two plates were coated by a capture antibody against 17β-HSD10  dissolved in carbonate buffer. Coating solution (246 ng of antibody per well) was applied and plates were incubated for 5 h at room temperature. 100 µl of samples or internal standards  according to the manufacturer’s instructions.Plate was coated by goat anti-rabbit IgG  and then by polyclonal rabbit anti-17β-HSD10 . 100 µl of redistilled water or of CSF samples were incubated in duplicates for 4 h. Subsequently, second primary antibody against cypD  and lately secondary antibody . After the subtraction of the blank, the values were compared to those from the internal standards  and expressed in percentage.+-EDTA, 10 mM Tris (pH 7.4) with 10% dimethyl sulfoxide in protein concentration of 5 mg/ml at − 80 °C. Before TEM analysis, the sample was tempered to room temperature and centrifuged at 14,000×g for 10 min. The pellet was fixed with 2.5% glutaraldehyde in 0.1 M cacodyladate buffer with 5 mM CaCl2 and embedded in 2% agar. The second fixation was done with 1% osmium tetroxide in 0.1 M cacodyladate buffer, serial dehydration and embedding into epoxy resin. The images were acquired with FEI Morgagni 268 transmission electron microscope operated at 80 kV equipped with Mega View III CCD camera (pixel size 6.45 × 6.45 µm).Sample of mitochondria was stored in 0.25 M sucrose, 0.5 mM KStatistical analysis was performed using BMDP statistical software. Parametric Welch test for global and Bonferroni adjusted t-test (separate variance) for pairwise comparisons (program 7D) were used . In addition, correlation analysis (program 6D) was applied. The equality of correlation coefficients (Rs) in two groups was examined using the test based on Fisher´s Z-transformation  . Sensitika, dissociation rate constant kd, and dissociation equilibrium constant Kd were determined as 2.0 × 105 M1s−1, 5.8 × 104 s−1, and 3.5 × 10−10 M, respectively. In order to confirm that the immobilized cypD is capable of interacting with its binding partners, we used Aβ 1-42 as a positive control  and reference (absence of cypD) channels. Figure ith cypD ). The coWe used also co-immunoprecipitation experiment to demonstrate the interaction of mitochondrial proteins 17β-HSD10 and cypD. Figure TEM documented high purity and intactness of our isolated mitochondria Fig. . IntactnResults of quantitative estimations are seen in Figs. Results of correlation analysis performed on data obtained from rat mitochondrial fraction revealed two significant negative correlations in WT control rats—the first between 17β-HSD10 and cypD levels  and the second between 17β-HSD10–cypD and 17β-HSD10–Aβ complexes . The correlation between 17β-HSD10 and cypD levels was not significant in Tg rats  and the result of Z–test supported the significant change between Tg and WT rats (p = 0.024). Analogously, the correlation between 17β-HSD10–cypD and 17β-HSD10–Aβ complexes was not significant in Tg animals , however, the result of Z-test indicated only borderline significance (p = 0.095).Analysis of CSF samples Table  revealedResults of correlation analysis performed on data from CSF samples revealed one significant negative correlation between age and Aβ 1–42  and one significant positive correlation between total-τ and phospho-τ levels  in the control group. The results of Z-test indicated a significant correlation loss in ADD  and FTLD groups  compared to the controls.In this work, we investigated the interaction between 17β-HSD10 and cypD in vitro using the SPR biosensor method and, for the first time, determined the kinetic parameters of this interaction. The interaction analysis revealed that 17β-HSD10 and cypD interact with high affinity and form a stable complex Fig. i and in With respect to particular levels of 17β-HSD10 and cypD, we have to note that our data obtained from quantitative ELISA did not reflect real concentrations occurring in the brain mitochondrial matrix of 11-month old WT rats since mitochondria were previously concentrated during the isolation process. However, the ratio of their size could be determined after recalculation of data to original mitochondrial fraction. Since the concentration of functional 17β-HSD10 homotetramers was around 0.135 µM (MW of human protein = 26.9 kDa) and that of cypD equaled 0.017 µM (MW of human protein = 18.9 kDA), it seems that the levels of tetrameric 17β-HSD10 are more than 7 times higher in the mitochondrial matrix when compared to those of cypD. Incidentally, it is in line with the fact that 17β-HSD10 is the essential protein with enzymatic as well as non-enzymatic functions necessary for structural and functional integrity of mitochondria , 33. It With respect to AD, the strength of the study is a well chosen animal model representing the human pathology. McGill-R-Thy1-APP rat model of AD is based on expression of human Aβ precursor protein carrying both the Swedish and Indiana mutations displays deficits in cognitive functions already in 3-month old rats  and cognOur experiments performed on the rat brain mitochondrial fraction isolated from the whole left hemisphere did not reveal significant differences between Tg and WT rats in 17β-HSD10 Fig. i or cypDOn the other hand, despite of unchanged levels of 17β-HSD10 Fig. i and cypIncidentally, although levels of 17β-HSD10–Aβ complexes are not significantly increased in mitochondria of McGill-R-Thy1-APP rats Fig. ii, the bAnd finally, our results revealed that CSF levels of 17β-HSD10–cypD complexes can be a very sensitive biomarker of AD including early disease stages (compare the very high sensitivity to MCI-AD 86.4% with that to ADD 92.9%). However, its specificity to AD pathology remains questionable when taking into account the similar results in FTLD patients. It seems that CSF levels of 17β-HSD10–cypD complexes reflect mitochondrial dysfunction in agreement with previous findings of mitochondrial dysfunction as a common feature of neurodegeneration . On the In this work, we used the SPR biosensor method to observe in vitro interaction of the two nucleus-encoded mitochondrial proteins, 17β-HSD10 and cypD, and, for the first time, determined kinetic parameters of the interaction. The interaction analysis revealed that the two proteins exhibit high affinity towards each other and form a stable complex. In brain mitochondria isolated from Tg animal model of AD (McGill-R-Thy1-APP rats) and in CSF samples of AD patients, we observed the significant drops in levels of 17β-HSD10–cypD complexes which could indicate the weakened function of 17β-HSD10 to regulate cypD in the mitochondrial matrix. CSF levels of 17β-HSD10–cypD complexes seem to be the very sensitive indicator of mitochondrial dysfunction observed in neurodegeneration but unfortunately not specific to AD pathology. We do not recommend it as the new biomarker of AD.


Background: To obtain wines with a lower percentage of alcohol, the simplest approach would be an earlier harvest of the grapes. However, this has implications for the wine composition and quality, due to the lack of phenolic maturity that these grapes may present. A technological innovation that could help in this situation could be the use of ultrasound in wineries. Methods: Grapes were harvested with two different ripening levels (25.4 °Brix and 29 °Brix), transported to the winery, and vinified. Also, a large-scale high-power ultrasound system was used to treat part of the less mature grapes just after crushing. These grapes were also vinified. The three different vinifications were skin-macerated for 7 days. The wine aroma compounds and physicochemical, chromatic, and sensory characteristics were analyzed at the time of bottling. Results: The wine made with the ultrasound-treated grapes showed very similar characteristics to the wine made with the more mature grapes, especially regarding total phenol and tannin content, but with an alcohol content 15% lower than the latter. Conclusions: The results indicate that this technology could be applied to grapes to favor the extraction of grape phenolic compounds, even when grape phenolic maturity is not complete, allowing the production of quality wines with a reduced alcohol content. Color is one of the most important quality attributes in red wine. It depends on the phenolic composition of the wine , and therefore, is closely bound to the grape phenolic composition.Grape phenolic compounds are mainly located in the skin (anthocyanins and tannins) and seeds (tannins), and are extracted to must during the crushing and maceration period. Although some enological techniques may help to extract these compounds , this exIn optimal situations, and when a variety is well adapted to a certain area, technological maturity  and phenolic and aromatic maturity  are reached at the same time. However, climate change is exerting a large influence on vine phenology and grape composition , and amoThe issue of high-alcohol wines is a big concern for winemakers, since it has potential implications for wine quality. Ethanol is sensorially important to wine, and is indispensable for the stability, aging, and organoleptic properties of wine —and therGiven all these issues, winemakers are really seeking different possibilities for obtaining high quality wines with a reduced alcohol content. Different approaches to reduce alcohol levels in wines have been proposed at all stages of the winemaking process, from the addition of unripe grape juice to finished wines ,18, to tIt could be, though, that one the easier solutions for wine alcohol reduction would be harvesting grapes with lower sugar content—if we can solve the problem of the low phenolic maturity that these grapes could present at this stage, and therefore, the difficulties in obtaining highly colored wines. Some enological techniques are focused in this issue, such as the use of maceration enzymes or prefermentative maceration techniques, as reviewed by Sacchi et al. , as wellAmong the novel technologies, high-power ultrasounds (HPUs) could be an interesting approach. This is a technology that has already been approved by the International Organization of Vine and Wine (OIV) in 2019 for its use in wineries. HPUs are generally comprised of frequencies between 20–40 kHz, with an energy level high enough to produce acoustic cavitation. This effect consists in the formation of tiny bubbles that grow until they reach a critical size that causes their implosion. During the implosion, remarkably high temperatures (circa 5000 K) and pressures (circa 2000 atm) are reached . When thTherefore, in enology HPUs could be used to(a)Improve the extraction of phenolic and aroma compounds from grapes ,26,27,28(b)2 by reducing microbial counts. In this way, Gracin et al. [Brettanomyces yeasts (89.1–99.7%) and lactic acid bacteria (71.8–99.3%), and Santos et al. [Reduce the use of SOn et al.  found ths et al.  review t(c)Saccharomyces strains coupled with ultrasound treatment to accelerate aging on lees, and studied their impacts on the polysaccharide release and on the organoleptic properties of red wine. Cacciola et al. [Age wines on lees: Kulkarni et al.  used nona et al.  tested t(d)Recover byproducts, such as phenolics from grape pomace ,34,35 or(e)_OH and _H radicals, that may undergo a range of subsequent reactions, including the generation of H2O2, and these highly oxidizing species can have a significant effect on both biological and chemical species in aqueous solution [Accelerate the reactions of aging: one other effect of cavitation is the production of highly reactive radical species, such as solution . The possolution . In thissolution  have prosolution  has statsolution  have staIn this work, the interest is focused on the effect that HPUs may have on facilitating the reduction of alcohol content of highly colored wines by the application of those HPUs to crushed grapes with lower sugar content.Monastrell red grapes were all harvested from one vineyard in the province of Murcia (Spain). The vineyard presented two differentiated areas with different maturation stages by a given date. Two separate harvests were done in these two areas, so we had two different batches of grapes from the same vineyard that differed in sugar content. Grapes were transported on the same day to the winery for their processing.2. The other half of the crushed grapes was not treated . The more mature grapes  were also destemmed and crushed . Small 50 kg, stainless steel tanks (per triplicate) were filled with both control grapes and with the ultrasound-treated crushed grapes. Must homogeneity in each tank was achieved by weighing the solid parts and the liquid must separately, and filling each 50 L vessel with the same quantity and proportion to assure the same solid/liquid ratio in each vessel. Total acidity was corrected, if necessary, to 5.5 g/L, and selected yeasts were added . The skin maceration time was 7 days for all the samples. Throughout the fermentation pomace contact period, the cap was punched down twice a day. At the end of this period, the wines were pressed in a 75 L pneumatic press. Free-run and press wines were combined and left at room temperature until the end of alcoholic fermentation. When the fermentation was finished, the wines were racked twice, cold stabilized at 2 °C for one month, and bottled. They were analyzed at the time of bottling. The less mature grapes  were destemmed and crushed. Half of the crushed grapes were treated  with a winery scale power ultrasound system  that can treat 400 kg of crushed grapes per hour. The system operated at 2500 W and 28 kHz frequency, with a power density of 8 W/cmThe wines were characterized by measuring the alcohol content, pH, total and volatile acidity, and acetic acid, according to European Community methods . Total aSpectrophotometric parameters: color intensity (CI) was calculated as the sum of absorbance at 620, 520, and 420 nm, and hue as the ratio between absorbance at 420 nm and absorbance at 520 nm. Total and polymeric anthocyanins were determined spectrophotometrically . Total tDetermination of tannins by the phloroglucinolysis method: Wine samples were prepared as described by Busse-Valverde et al.  from an For the isolation of major volatile compounds by solid-phase microextraction (SPME), a divinylbenzene–carboxen–polydimethylsiloxane (DVB/CAR/PDMS) fiber was used. It was conditioned before the first use by insertion into the gas chromatograph injector, as recommended by the manufacturer. For the analysis of wine volatile compounds, 10 mL of wine were added to a 20 mL headspace vial. Four grams of sodium chloride and 50 µL of the internal standard (125 µL/L of 2-octanol in absolute ethanol) were added to the same vial. The vial was sealed and loaded onto a Gerstel auto-sampling device , and the analysis was conducted using an Agilent 6890N gas chromatograph coupled to an Agilent 7890B single quadrupole mass spectrometer . The conditions of the microextraction procedure, the gas chromatograph, and the mass spectra conditions can be found in Gómez-Plaza et al. . Peak idWines were subjected to sensory evaluation using a descriptive test. Prior to the sensory analysis, the wine from the three different replications for each experience was pooled to have a representative sample, and to avoid differences among the replications. Ten staff members with experience in wine sensory analysis and interest in the project were selected for the sensory analysis.Forty mL of each wine was poured 30 min before evaluation. Glasses were coded and presented to judges in a sensory room that was kept at 20 °C and free of unusual odors. Each panelist sat in an individual isolated booth illuminated with white light. The intensity of each attribute was rated on a scale of zero to five, with a score of zero indicating that the descriptor was not perceived. Data from all the judges for all samples were used in the analysis.The analysis of variance and the principal component analysis were carried out using the statistical package Statgraphics Centurion XVI.3 .Many of the studies about the characteristics of wines made from grapes with different ripening degrees were done on grapes sampled at different times during ripening. However, it is also well known that, at any given point, the physiological characteristics of grape berries in a vineyard are very heterogeneous . This isWhen comparing C14 with US14, no significant differences were found in any of the physico-chemical properties, except for the content in methanol. Methanol is produced before and during alcoholic fermentation from the hydrolysis of pectins by pectinase enzymes (such as pectin methylesterase), which are naturally present in the fruit. More methanol is produced when must is fermented on grape skins; hence, there is generally more methanol in red wines than in rosé or white wines . The higBotrytis cinereainereal, Penicillium, Aspergillus, and Mucor, and bacteria, such as Acetobacter and Gluconobacter, and their presence in must and wines is related to the level of grape infection, which is facilitated by climatic factors like moisture and rainfall, as well as by physiological factors like ripening stage [If we compared the C14 and US14 with C16, the differences were as expected. The alcohol content of C16 was almost two units higher than C14 and US14. Differences in residual sugars and pH were small, but total acidity and volatile acidity was higher in C16, with no differences in tartaric acid content (acidity was corrected in the must when necessary). Gluconic acid increased in the wines made from the more mature grapes. Gluconic acid is a product originated by fungi, such as ng stage . Therefong stage . The maxEthanal was also controlled to determine if the use of HPUs at the beginning of the winemaking process could, somehow, affect the evolution of vinification and the presence of oxidation markers. This molecule is produced by ethanol oxidation and although it may, at low concentrations, contribute to red wine color evolution during aging , an exceWhen comparing the C14 and US14 wine chromatic parameters, the positive effect of the application of HPUs to the crushed grapes was clearly observed, with the US14 wine presenting significantly higher values for all the chromatic parameters measured than the C14 wine, except for total anthocyanins, which did not differ from C14 wine. Moreover, US14 wine did not statistically differ in any chromatic parameter from C16 wine. Our previous studies in the application of HPUs to crushed grapes have shown the increase in chromatic parameters due to HPUs application and its usefulness for reducing maceration time in wineries . SimilarWhen comparing C14 and US14 wine, important differences were observed. The concentration of tannins was much higher in the wine elaborated with sonicated grapes. The lower value of mDP, together with the higher value of epicatechin gallate subunit (ECG) in US14 wine indicated a higher extraction of tannins from seeds. Furthermore, the concentration of EGC was similar in C14 and US14 and the most important difference was the concentration of ECG. Up until now, there are no studies where the effect of HPUS on seed integrity has been reported but the results pointed to an effect of HPUS in the easiness of seed tannin extraction. Comparing US14 and C16 wines, US14 wine presented higher tannin concentration and a lower mDP than C16 wine, reiterating the positive effect of HPUS in tannin extraction, which may help to ensure a high wine color stability during storage. The application of HPU technology to crushed grapes and its effect on the wine’s volatile compounds has been less studied than its effect on phenolic compounds. Bautista-Ortin et al.  studied Esters, as one of the most important odorants in wines, provide abundant floral and tropical fruity aromas . The oriThe higher presence of both types of esters implies that the sum of esters was higher in C16 wine. These results were also reported by Bindon et al. , who fouThe production of higher alcohols is linked to the amino acid metabolism by yeasts, and the alcohol deshydrogenase enzymes in fruit and yeasts are the responsible for catalyzing the reduction of aliphatic aldehydes to alcohols . We founComparing C14 and US14 wine, no difference in the sum of total alcohols could be observed although some differences in individual alcohols were found, such as higher concentrations of 3-methyl-1-butanol, 1-heptanol, or 2-phenylethanol, and lower concentrations of hexanol in C14 wine. Although higher alcohols can contribute to a positive effect on wine aroma if they are not present at very high concentrations , Zhang eConcentrations of linear fatty acids decreased in the wine made with the more mature grapes, except for the concentration of acetic acid, which increased in these wines, as would be expected, because concentrations of sugars also increased. These acids are important in the wine aroma balance, although they may impart an unpleasant fatty odor, and even a rancid smell in wine, when present at high concentrations. Although a higher concentration of fatty acids has been described in must from mature grapes , the redAmong other compounds, benzaldehyde  can be associated with defective wines . It is pThe results up to now point to large differences in the phenolic compounds and chromatic characteristics of wine due to the use of ultrasounds in the winery, while variations in wines’ volatile compounds were not so evident. However, the most important tool researchers have for evaluating if the observed changes in phenolic and volatile compounds due to ultrasounds will have any effect on consumers’ appreciation is the sensory analysis. Wines were subjected to a descriptive sensory analysis, and Mouthfeel intensity and body scores were significantly higher in C16 wine , and no differences in mouthfeel quality, equilibrium, or persistence could be detected among wines. Bitterness was higher in C16 than in C14 or US14. The greater bitter sensation imparted by C16 wine has to be attributed to the higher alcohol content. Noble  reportedA principal component analysis was conducted using all the measured chromatic and phenolic parameters, together with the sum of major aroma compounds and sensory scores as variables. This analysis reduced the information provided by all the measured variables to two principal components, and explained 78% of the variability of the data . The objThe different weights of the variables in achieving the sample separation can be seen in In conclusion, the results showed that the wine obtained with ultrasound-treated grapes presented chromatic characteristics that did not differ to those of a wine obtained from more mature grapes, and reached the highest scores in aroma and mouthfeel quality descriptors in a sensory analysis. The use of ultrasound technology, being a clean, eco-friendly, and energetically very efficient technology—and not less importantly, being an authorized practice in wineries—could be an interesting option for obtaining wines with similar color intensity and sensorial quality parameters as wines obtained from more mature grapes, but with a lower alcohol content.