[Application of raman spectroscopy in the male reproductive system].

Current diagnostic techniques for male infertility primarily require invasive testing of sperm. Clinically, there is a need for a reliable, non-invasive analysis method that provides precise information about sperm quality without compromising sperm cell integrity. Raman spectroscopy, utilizing the inelastic scattering spectra of light, offers a rapid, simple, repeatable, and non-destructive approach for both qualitative and quantitative analysis, gaining widespread application in medicine. This paper reviews the fundamental characteristics of Raman spectroscopy and its applications in the male reproductive system.

Integrated evidence reveals a new species of Cosmocerca (Ascaridomorpha: Cosmocercoidea) from the Asiatic toad Bufo gargarizans Cantor (Amphibia: Anura).

Species of Cosmocerca Diesing, 1861 (Ascaridomorpha: Cosmocercoidea), are common nematode parasites of amphibians. In the present study, a new species of Cosmocerca, namely C. simile n. sp., was described using light and scanning electron microscopy, and sequencing different nuclear and mitochondrial genetic markers (i.e. small ribosomal DNA (18S), large ribosomal DNA (28S), internal transcribed spacer (ITS) and cytochrome c oxidase subunit 1 (cox1)). Cosmocerca simile n. sp. differs from its congeners based on body size, morphology and number of plectanes, relative length of spicules and gubernaculum and spicules to total body length and morphology and length of tail. Molecular analysis showed no nucleotide polymorphisms among different individuals of the new species regarding nuclear DNA. Very low intraspecific nucleotide variation (0.52-0.78%) was detected in cox1 mtDNA. In contrast, the level of interspecific nucleotide variation between C. simile n. sp. and its congeners were distinctly higher (2.74-18.1% in the partial ITS region and 10.2-13.5% in the partial cox1 region, respectively) than that of intraspecific variation. Phylogenetic analyses using maximum likelihood (ML) inference based on the partial ITS and cox1 sequence data both supported the new species to be a member of the genus Cosmocerca, and formed a sister relationship to C. japonica. The newly obtained genetic data are important for further studies of DNA-based taxonomy, population genetics and phylogenetics of the Cosmocercoidea.

Molecular Phylogeny and Dating Reveal a Terrestrial Origin in the Early Carboniferous for Ascaridoid Nematodes.

Ascaridoids are among the commonest groups of zooparasitic nematodes (roundworms) and occur in the alimentary canal of all major vertebrate groups, including humans. They have an extremely high diversity and are of major socio-economic importance. However, their evolutionary history remains poorly known. Herein, we performed a comprehensive phylogenetic analysis of the Ascaridoidea. Our results divided the Ascaridoidea into six monophyletic major clades, i.e., the Heterocheilidae, Acanthocheilidae, Anisakidae, Ascarididae, Toxocaridae, and Raphidascarididae, among which the Heterocheilidae, rather than the Acanthocheilidae, represents the sister clade to the remaining ascaridoids. The phylogeny was calibrated using an approach that involves time priors from fossils of the co-evolving hosts, and dates the common ancestor of the Ascaridoidea back to the Early Carboniferous (approximately 360.47-325.27 Ma). The divergence dates and ancestral host types indicated by our study suggest that members of the Ascaridoidea first parasitized terrestrial tetrapods, and subsequently, extended their host range to elasmobranchs and teleosts. We also propose that the fundamental terrestrial-aquatic switches of these nematodes were affected by changes in sea-level during the Triassic to the Early Cretaceous.

Morphological and molecular evidence for a new species of the genus Cosmocercoides Wilkie, 1930 (Ascaridida: Cosmocercidae) from the Asiatic toad Bufo gargarizans Cantor (Amphibia: Anura).

A new cosmocercid species, Cosmocercoides qingtianensis sp. n., collected from the intestine of the Asiatic toad Bufo gargarizans Cantor (Amphibia: Anura) is described using integrated approaches, including light and scanning electron microscopy, and sequencing and analyzing the ribosomal [small ribosomal DNA (18S) and internal transcribed spacer (ITS)] and mitochondrial [cytochrome c oxidase subunit 1 (cox1)] target regions, respectively. The new species can be distinguished from its congeners by the combination of the following morphological characters, including the large body size, the presence of lateral alae and somatic papillae in both sexes, the length of spicules, the particular morphology and length of gubernaculum, the number, arrangement and morphology of caudal rosettes, the presence of large medioventral precloacal papilla and the long tail. Our molecular analysis revealed the level of intraspecific genetic variation of C. qingtianensis sp. n. distinctly lower than that of the interspecific genetic variation in the ITS and cox1 regions. However, there are some overlaps in the range of intra- and interspecific 18S sequence divergence between the new species and some closely related species. The results of molecular analysis supported the validity of the new species based on the morphological observations. The 18S, ITS, and cox1 regions of C. pulcher collected from Bufo japonicus formosus in Japan were also sequenced and analyzed. The results showed a low level of intraspecific genetic variation in 18S and ITS regions (0-0.12% and 0-0.23% nucleotide differences, respectively), but a relatively high level of intraspecific genetic variation in cox1 region (0.78-4.69% nucleotide differences). In addition, it seems more powerful and practical to use the cox1 region as a genetic marker for the accurate identification and differentiation of species of Cosmocercoides than the 18S and ITS regions, especially for the closely related species.

Morphological and molecular study of Longicollum pagrosomi Yamaguti, 1935 (Acanthocephala: Pomphorhynchidae) from the barred knifejaw Oplegnathus fasciatus (Temminck & Schlegel) (Perciformes: Oplegnathidae) in the East China Sea.

Longicollum pagrosomi Yamaguti, 1935 (Acanthocephala: Pomphorhynchidae) collected from the barred knifejaw Oplegnathus fasciatus (Temminck & Schlegel) (Perciformes: Oplegnathidae) in the East China Sea (off Zhoushan Islands) was studied using light and scanning electron microscopy. The SEM observations revealed for the first time the presence of about 28 well-developed sensory papillae arranged in a circle on the copulatory bursa. In addition, L. pagrosomi was characterised using molecular methods by sequencing of the internal transcribed spacer (ITS) of the ribosomal DNA based on the newly collected material. Longicollum pagrosomi is the first species of the genus with the ITS region sequenced for the purpose of species identification. These new morphological and molecular data contributed to a reliable and accurate specific identification and differentiation of species.

Morphological and molecular identification of Habronema spp. (Nematoda: Habronematidae) from donkeys in Xinjiang, China, and notes on the taxonomical status of Habronema majus (Creplin, 1849) and H. microstoma (Schneider, 1866).

Habronematid nematodes were collected from the stomachs of donkeys, Equus asinus L., in the Tarim Basin, Xinjiang, China. After examination by light and scanning electron microscopy, Habronema muscae (Carter, 1861) and H. majus (Creplin, 1849) were identified. The morphology of our specimens representing H. muscae (Carter, 1861) agreed well with previous redescriptions in the shape of the lateral lips, origin of the lateral alae, ratio of left and right spicules, and number and arrangement of caudal papillae. However, H. majus (Creplin, 1849) differs from H. microstoma (Schneider, 1866) in the arrangement of the caudal papillae in the male. Moreover, molecular analysis also showed interspecific differences of 26.2-28.2% in ITS2 and 8.6-8.9% in cox1 between H. majus and H. microstoma, a divergence much higher than the known intraspecific variation of Habronema spp. (6.6-8.7% in ITS2; 0.2-2.2% in cox1). The results indicate that both H. microstoma (Schneider, 1866) and H. majus (Creplin, 1849) are valid species.

Neoascarophis sinensis n. sp. (Nematoda: Cystidicolidae), a parasite of Conger myriaster (Brevoort) (Anguilliformes: Congridae) in Chinese waters.

Neoascarophis sinensis n. sp. collected from the whitespotted conger Conger myriaster (Brevoort) (Anguilliformes: Congridae) in the Yellow and East China Seas, is described using both light and scanning electron microscopy. The new species is characterised mainly by the body size (8.5-10.5 mm in the males, 9.5-14.0 mm in the females), the location of the vulva (near equatorial region of the body), the non-bifurcate deirids, the lengths of the vestibule (40-50 µm in the males, 30-60 µm in the females) and glandular oesophagus (2.5-3.1 mm in the males, 3.1-3.5 mm in the females) and the morphology and length of the spicules (left spicule 400-410 µm, right spicule 130-150 µm). Neoascarophis sinensis n. sp. is the first species of Neoascarophis Machida, 1976 reported from the anguilliform fish and is also the only species of this genus found in the Chinese waters.

An annotated catalogue of the ascaridoid nematode parasites of Chinese vertebrates.

A catalogue, based on both examined specimens and the published literature, of all the ascaridoid nematodes recorded in China is presented. A total of 95 recognised species, representing 26 genera in five families, are reported. Detailed information on the type-host, type-locality, original reference, synonyms, annotated subsequent references of taxonomic importance, other host records, site of infection, location of type-specimens and distribution are listed for each recognised species. Additional comments on the taxonomic status of some species are also given. Moreover, some nomenclatural changes are proposed: (i) Toxascaris selenarctis Wang, 1965 and T. ailuri Wu, He & Hu, 1987 are placed in synonymy with Baylisascaris transfuga (Rudolphi, 1819); (ii) Raphidascaris lophii Wang & Wu, 1991 is a secondary homonym of R. lophii (Wu, 1949) and a replacement name, R. wangi nom. nov., is proposed for the former species; (iii) Aliascaris aetoplatea Luo, 2001 is transferred to Terranova Leiper & Atkinson, 1914, as T. aetoplatea (Luo, 2001) n. comb., and should be considered a species inquirenda; (iv) Ophidascaris orientalis (Wang, 1965) is resurrected as a valid species; (v) Phocascaris longispiculum Wang & Wu, 1991 and Ophidascaris agkistrodontis Wang, 1979 are treated as incertae sedis; and (vi) Hysterothylacium sauridae Li, Xu & Zhang, 2008 is listed as a nomen nudum.

Morphological study of Ophidascaris excavata Hsü & Hoeppli, 1931 (Ascaridida: Ascarididae) from Gloydius brevicaudus (Stejneger) (Reptilia: Viperidae).

Ophidascaris excavata Hsü & Hoeppli, 1931 is a poorly known ascaridid parasite reported from the short-tailed pit viper Gloydius brevicaudus (Stejneger) (Reptilia: Viperidae) in China. In the present paper, the detailed morphology of this nematode was studied using light and scanning electron microscopy (SEM) based on newly collected material. The results revealed several important, but previously unreported, morphological features, including the presence of one pair of small, finger-like prolongations on each lip, narrow cervical alae beginning well posterior to the base of the ventrolateral lips and the second pair of postcloacal ventro-lateral papillae being double; in addition, there is no intestinal caecum. These supplementary morphological and morphometric data, especially the detailed morphological features obtained herein under SEM, would help us to understand the relationships of O. excavata with its congeners and enable us to diagnose this species more accurately.

MicroRNA profile of tumorigenic cells during carcinogenesis of lung adenocarcinoma.

To obtain microRNA (miRNA) profile and clarify their biological function in tumorigenic Sca-1(+) CD34(+) cells during carcinogenesis of lung adenocarcinoma. After intranasal infection with recombinant Adeno-Cre viruses (AdV-Cre), lung adenocarcinoma was identified pathologically in Lox-stop-lox Kras (LSL-Kras) G12D mice. Sca-1(+) CD34(+) cells were sorted by flow cytometry and tested for tumor-initiating ability, self-renewal and tumorigenicity. MiRNA profiles were obtained using microarray and further confirmed by real-time RT-PCR (qRT-PCR). MiRNA functions were predicted bioinformatically, and miR-294 function was verified to explore its role in tumor migration and invasion. Lung adenocarcinoma was induced in LSL-Kras G12D mice within 30 days. In vivo, the tumorigenicity of Sca-1(+) CD34(+) cells was 25 times stronger than Sca-1(-) CD34(-) cells. During tumorigenesis of lung adenocarcinoma, the expression of 145 miRNAs in Sca-1(+) CD34(+) cells increased and 72 miRNAs decreased (P < 0.01). Four successively up-regulated miRNAs (miR-15a*, miR-203, miR-294 and miR-295*) and three successively down-regulated ones (miR-19b, miR-483 and miR-615-5p) were identified. Among them, miR-294 could constitutively bind to 3'-UTR of matrix metalloproteinase 3 (MMP3), and down-regulate MMP3 protein expression. MiR-294 also significantly inhibited migration and invasion of Lewis lung cancer cells. MiRNAs are characteristically expressed in tumor-initiating Sca-1(+) CD34(+) cells of lung adenocarcinoma, and may play important roles during the carcinogenesis of lung adenocarcinoma.

Essential role of miR-200c in regulating self-renewal of breast cancer stem cells and their counterparts of mammary epithelium.

BACKGROUND: Breast cancer stem cells (BCSCs) have been reported as the origin of breast cancer and the radical cause of drug resistance, relapse and metastasis in breast cancer. BCSCs could be derived from mutated mammary epithelial stem cells (MaSCs). Therefore, comparing the molecular differences between BCSCs and MaSCs may clarify the mechanism underlying breast carcinogenesis and the targets for gene therapy. Specifically, the distinct miRNome data of BCSCs and MaSCs need to be analyzed to find out the key miRNAs and reveal their roles in regulating the stemness of BCSCs. METHODS: MUC1(-)ESA(+) cells were isolated from normal mammary epithelial cell line MCF-10A by fluorescence-activated cell sorting (FACS) and tested for stemness by clonogenic assay and multi-potential differentiation experiments. The miRNA profiles of MaSCs, BCSCs and breast cancer MCF-7 cells were compared to obtain the candidate miRNAs that may regulate breast tumorigenesis. An miRNA consecutively upregulated from MaSCs to BCSCs to MCF-7 cells, miR-200c, was chosen to determine its role in regulating the stemness of BCSCs and MaSCs in vitro and in vivo. Based on bioinformatics, the targets of miR-200c were validated by dual-luciferase report system, western blot and rescue experiments. RESULTS: In a 2-D clonogenic assay, MUC1(-)ESA(+) cells gave rise to multiple morphological colonies, including luminal colonies, myoepithelial colonies and mixed colonies. The clonogenic potential of MUC1(-)ESA(+) (61.5 ± 3.87 %) was significantly higher than that of non-stem MCF-10A cells (53.5 ± 3.42 %) (P < 0.05). In a 3-D matrigel culture, MUC1(-)ESA(+) cells grew into mammospheres with duct-like structures. A total of 12 miRNAs of interest were identified, 8 of which were upregulated and 4 downregulated in BCSCs compared with MaSCs. In gain- and lost-of-function assays, miR-200c was sufficient to inhibit the self-renewal of BCSCs and MaSCs in vitro and the growth of BCSCs in vivo. Furthermore, miR-200c negatively regulated programmed cell death 10 (PDCD10) in BCSCs and MaSCs. PDCD10 could rescue the tumorigenesis inhibited by miR-200c in BCSCs. DISCUSSION: Accumulating evidence shows that there is a milignant transformation from MaSCs into BCSCs. The underlying mechanism remains unclear. In present study, miRNA profiles between MaSCs and BCSCs were obtained. Then miRNA-200c, downregulated in both MaSCs and BCSCs, were verified as anti-oncogene, and played essential role in regulating self-renewal of both kinds of stem-like cells. These findings reveal a novel insights of breast tumorigenesis. CONCLUSIONS: PDCD10 is a target gene of miR-200c and also a possible mechanism by which miR-200c plays a role in regulating the stemness of BCSCs and MaSCs.

Dujardinascaris gigantea sp. n. (Nematoda: Ascaridida) from the critically endangered crocodile Alligator sinensis Fauvel (Reptilia: Crocodylia).

The Chinese alligator Alligator sinensis Fauvel (Reptilia: Crocodylia) is considered as one of the most critically endangered species of the 23 extant crocodiles. However, our knowledge of the helminth parasites of this rare animal is completely lacking. During a helminthological survey of reptiles in China, we found a new ascaridoid nematode, Dujardinascaris gigantea sp. n. from A. sinensis. The morphology of D. gigantea sp. n. was studied using light and scanning electron microscopy. The new species was also characterised using molecular methods by sequencing and analysing the small ribosomal DNA (18S) and the second internal transcribed spacer (ITS-2).

Porrocaecum parvum n. sp. and P. reticulatum (Linstow, 1899) (Nematoda: Ascaridoidea) from birds in China.

Porrocaecum parvum n. sp. is described from the grey-faced buzzard Butastur indicus (Gmelin) (Accipitriformes: Accipitridae) in China. The new species differs from its congeners in having well-developed cervical alae, small interlabia and very short intestinal caecum (0.34 mm long, representing 11.9% of oesophageal length) and in the number and arrangement of the caudal papillae (29 pairs in total, arranged as follows: 21 pairs precloacal, single double pair paracloacal and seven pairs postcloacal) and in the morphology of the male tail. In addition, Porrocaecum reticulatum (Linstow, 1899), collected from the purple heron Ardea purpurea L., the grey heron A. cinerea L. and the little egret Egretta garzetta (L.) (Pelecaniformes: Ardeidae) in China, was also studied using light and, for the first time, scanning electron microscopy. Previously unreported and erroneous morphological features of taxonomic significance are revealed, including the presence of narrow cervical alae, single pair of small, submedial pores and single, short medial ditch on each lip, interlabia with very pointed anterior prolongation, single medio-ventral precloacal papilla on anterior cloacal lip and double paracloacal papillae slightly posterior to cloaca.

Morphology, ultrastructure and molecular characterisation of Spiroxys japonica Morishita, 1926 (Spirurida: Gnathostomatidae) from Pelophylax nigromaculatus (Hallowell) (Amphibia: Ranidae).

Gnathostomatid nematodes identified morphologically as Spiroxys japonica Morishita, 1926 were collected from the dark-spotted frog Pelophylax nigromaculatus (Hallowell) (Amphibia: Ranidae) in China. Light and scanning electron microscopy were used to study the morphology of this species in detail. Previously unreported morphological features are revealed and others corrected. In addition, adult nematodes of S. japonica collected from P. nigromaculatus and Spiroxys hanzaki Hasegawa, Miyata & Doi, 1998 collected from Andrias japonicus (Temminck) (Caudata: Cryptobranchidae) in China and Japan, respectively, and the third-stage larva of S. japonica collected from Lithobates catesbeianus (Shaw) (Anura: Ranidae) in Japan, were characterised using molecular methods by sequencing and analysing ribosomal [large ribosomal DNA (18S) and internal transcribed space] and mitochondrial [cytochrome c oxidase subunit 1] target regions, respectively. The new morphological and genetic data contributes to a more accurate diagnosis of this hitherto little known nematode genus.

Occurrence of Hysterothylacium and Anisakis nematodes (Ascaridida: Ascaridoidea) in the tanaka's snailfish Liparis tanakae (Gilbert & Burke) (Scorpaeniformes: Liparidae).

The tanaka's snailfish Liparis tanakae (Gilbert & Burke) (Scorpaeniformes: Liparidae) is an economically important marine fish species in China. However, the helminth parasites of this fish are still poorly known. During a helminthological survey of Chinese marine fishes from 2011 to 2012, we revealed that L. tanakae was heavily infected with third-stage larvae and adults of ascaridoid nematodes (total prevalence 100% and mean intensity 82.3 nematodes per fish). Four species of third-stage larvae Hysterothylacium liparis Li, Xu & Zhang, 2007, H. aduncum (Rudolphi, 1802), Hysterothylacium fabri (Rudolphi, 1819), and Anisakis pegreffii (Campana-Rouget & Biocca, 1955) and a single species of adults H. liparis were differentiated and identified by morphological and molecular methods. The detailed morphology of the four species of third-stage larvae was also studied using light microscopy and scanning electron microscopy. The morphological and molecular characterization of the third-stage larvae of H. liparis was reported. Liparis tanakae represents a new host record for A. pegreffii and H. fabri. In addition, a new name, Hysterothylacium zhoushanense nom. nov. was also given to Hysterothylacium zhoushanensis Li, Liu & Zhang, 2012 to make the latinized specific epithet agree with this neuter generic name.

Ophidascaris wangi sp. n. and O. najae (Gedoelst, 1916) (Ascaridida: Ascaridoidea) from snakes in China.

Ophidascaris wangi sp. n. collected from the king rat snake Elaphe carinata (Günther) (Serpentes: Colubridae) in China is described using both light and scanning electron microscopy. The new species differs from its congeners in the presence of narrow lateral alae originating a short distance posterior to the base of the ventrolateral lips, its relatively long oesophagus (3.57-4.54 mm long, representing 6.6-7.6% of body length), its short spicules (1.89-2.14 mm long, representing 3.9-4.3% of body length), the number and arrangement of caudal papillae (49-57 pairs in total, arranged as follows: 43-51 pairs precloacal, 2 pairs joined paracloacal and 4 pairs postcloacal), the presence of a particular papilliform medioventral, postcloacal ornamentation and the morphology of the eggs and tip of the female tail. In addition, Ophidascaris najae (Gedoelst, 1916), collected from the king cobra Ophiophagus hannah Cantor (Serpentes: Elapidae) in China, is also redescribed. The morphology of the cervical papillae, labial denticles and phasmids of the female is described for the first time.

Morphological and molecular evidence for a new species of the genus Dichelyne Jägerskiöld, 1902 (Ascaridida: Cucullanidae) from marine perciform fishes in the South China Sea.

A new cucullanid nematode, Dichelyne (Dichelyne) breviculus n. sp., collected from the intestine of the goatee croaker Dendrophysa russelii (Cuvier) (Perciformes: Sciaenidae), the burrowing goby Trypauchen vagina (Bloch & Schneider) and the tropical sand goby Acentrogobius caninus (Valenciennes) (Perciformes: Gobiidae) in the South China Sea, is described using both light and scanning electron microscopy. The new species differs from its congeners in the size of body (2.16-2.96 mm in male), the position of the excretory pore and deirids, the length of the spicules (0.90-1.32 mm, representing 32.4-51.9% of body length), the arrangement of the caudal papillae and the morphology of the tail. In addition, in order to primarily assess the validity of the new species genetically, the specimens of D. breviculus n. sp. collected from the three different hosts were also characterised using molecular methods by sequencing and analysing ribosomal [small ribosomal subunit (18S rDNA) and internal transcribed spacer (ITS)] and mitochondrial [cytochrome c oxidase subunit 1 (cox1)] target regions. The molecular analyses support the validity of the new species based on the morphological observations.

Morphological variability and molecular characterisation of Dichelyne (Cucullanellus) pleuronectidis (Yamaguti, 1935) (Ascaridida: Cucullanidae) from the flatfish Pleuronichthys cornutus (Temminck & Schlegel) (Pleuronectiformes: Pleuronectidae) in the East China Sea.

Cucullanid nematodes identified morphologically as Dichelyne (Cucullanellus) pleuronectidis (Yamaguti, 1935) were collected from the ridged-eye flounder Pleuronichthys cornutus (Temminck & Schlegel) (Pleuronectiformes: Pleuronectidae) in the East China Sea. Their examination using light microscopy and, for the first time, scanning electron microscopy, revealed several important, but previously unreported morphological features and the presence of remarkable morphological differences in the intestinal caecum and deirids among some individuals. Consequently, specimens of D. pleuronectidis were characterised using molecular methods by sequencing and analysing the internal transcribed spacer (ITS) of the ribosomal DNA to test whether the present material with broad range of morphological variability, represents a complex of sibling species or a single species. The results of molecular analyses proved that the differences in the intestinal ceacum and deirids should be considered as intraspecific variation and that the nematode material collected from P. cornutus in the East China Sea represented a single species, D. pleuronectidis. These new morphological and genetic data contributed to an accurate diagnosis of this hitherto insufficiently known nematode and also indicated that a more rigorous study based on morphological and genetic data with broader representation of the Cucullanidae is required to assess whether the traditionally used diagnostic character of absence or presence of intestinal ceacum is of generic importance in distinguishing Dichelyne and Cucullanus.

Analysis of Nonhomologous End Joining and Homologous Recombination Efficiency in HEK-293T Cells using GFP Based Reporter Systems.

DNA double-strand breaks (DSBs) represent the most perilous DNA lesions, capable of inducing substantial genetic information loss and cellular demise. In response, cells employ two primary mechanisms for DSB repair: nonhomologous end joining (NHEJ) and homologous recombination (HR). Quantifying the efficiency of NHEJ and HR separately is crucial for exploring the relevant mechanisms and factors associated with each. The NHEJ assay and HR assay are established methods used to measure the efficiency of their respective repair pathways. These methods rely on meticulously designed plasmids containing a disrupted green fluorescent protein (GFP) gene with recognition sites for endonuclease I-SceI, which induces DSBs. Here, we describe the extrachromosomal NHEJ assay and HR assay, which involve co-transfecting HEK-293T cells with EJ5-GFP/DR-GFP plasmids, an I-SceI expressing plasmid, and an mCherry expressing plasmid. Quantitative results of NHEJ and HR efficiency are obtained by calculating the ratio of GFP-positive cells to mCherry-positive cells, as counted by flow cytometry. In contrast to chromosomally integrated assays, these extrachromosomal assays are more suitable for conducting comparative investigations involving multiple established stable cell lines.

FAM21 interacts with Ku to promote the localization of WASH to DNA double strand break sites.

Cytoplasmic FAM21 works as a guiding protein in Wiskott-Aldrich Syndrome Protein and SCAR Homolog (WASH) complex by linking WASH complex to endosomes through its interaction with retromer. Recently, we have reported that nuclear WASH localizes to DNA double strand break (DSB) sites to promote DNA repair through non-homologous end-joining (NHEJ). However, whether FAM21, the close partner of WASH, is involved in the nuclear WASH localization and DNA repair remains to be clarified. Here, we show that FAM21 interacts with Ku and the interaction between C-terminal FAM21 and Ku is essential for its recruitment to DSB sites. Moreover, FAM21 depletion led to decreases in WASH recruitment to damaged DNA and repair capacity upon DNA damage. Taken together, these results reveal that FAM21 promotes DNA repair by orchestrating the recruitment of WASH to DSB sites, providing a mechanistic insight into WASH-dependent DNA DSB repair.