In terms of mode of action, these antipsychotics are either antagonist or partial agonist at the above-mentioned receptors. Although D2Rs and D3Rs possess high degree of homology in their molecular structure, have common signaling pathways and similar in vitro pharmacology, they have different in vivo pharmacology and therefore behavioral roles. The aim of this review, with summarizing preclinical and clinical evidence is to demonstrate that while currently used antipsychotics display substantial in vitro affinity for both D3Rs and D2Rs, only very few can significantly occupy D3Rs in vivo. The relative importance of the level of endogenous extracellular dopamine in the brain and the degree of in vitro D3Rs receptor affinity and selectivity as determinant factors for in vivo D3Rs occupancy by antipsychotics, are also discussed.Dysfunctions of the dopaminergic system are believed to play a major role in the core symptoms of schizophrenia such as positive, negative, and cognitive symptoms. The first line of treatment of schizophrenia are antipsychotics, a class of medications that targets several neurotransmitter receptors in the brain, including dopaminergic, serotonergic, adrenergic and/or muscarinic receptors, depending on the given agent. Although the currently used antipsychotics display  Currently used antipsychotics display medium-to-high in vitro affinity for D2R as well as D3R, and high correlation can be demonstrated between their affinities for these receptors. This is not surprising considering the high structural homology, and the in vitro functional and pharmacological similarities of the two receptors. On the other hand, significant differences have been demonstrated in their in vivo pharmacology and behavioral roles. All currently used antipsychotics, in agreement with their in vitro D2R affinity, show significant in vivo brain D2R occupancy at their antipsychotic effective doses. However, despite their substantial in vitro D3R affinity, not all antipsychotics demonstrated in vivo D3R occupancy in animals or in humans. Here, a review is given on the data available for the in vitro affinity for D2Rs and D3Rs and a hypothesis is provided as to why a group of antipsychotics do not show significant in vivo brain D3R occupancy despite their notable in vitro D3R affinity.It is widely accepted that dysfunction of the dopaminergic neurotransmitter system plays a major role in the pathophysiology of schizophrenia. The primary pharmacotherapy of schizophrenia involves the use of antipsychotics, a group of drugs representing great heterogeneity in their chemical structure, pharmacological and functional profile, as well as clinical efficacy. At present, all available antipsychotics display affinity for DSchizophrenia is one of the most serious and debilitating psychiatric disorder affecting about 1% of the population disregarding economic, social, or cultural background of the society –4.The early, so called “dopamine hypothesis” stated that low prefrontal dopamine activity would cause “deficit symptoms” whereas enhanced activity in mesolimbic dopamine system would be in the background of the positive symptoms . In factRecognition of the neuroleptic action of chlorpromazine in 1952 represented a breakthrough in the drug treatment of schizophrenia . ChlorprAt the time of their discovery, the main mechanism of action of the first-generation antipsychotics was believed to be mediated by their actions on the monoaminergic system. Carlsson and Lindquist demonstrated that haloperidol and chlorpromazine increased monoamine turnover in the rat brain and these changes were attributed to the monoamine receptor antagonism action of these compounds . Van Ros1 receptors (D1Rs), D2R and serotonin 5-HT2 receptors (5-HT2R). They found that the 5-HT2R/D2R affinity ratio was the most useful means of differentiation from the typical antipsychotics. They demonstrated that compounds displaying 5-HT2R/D2R affinity ratio of 1.12 or higher were the ones showing the atypical characteristics (2A receptor (5-HT2AR) affinity that would be similar or higher than that for the D2R. The quest for compounds with D2R/5-HT2AR affinity led to discovery of risperidone, asenapine, olanzapine, quetiapine, ziprasidone, blonanserin and lurasidone, collectively classified as atypical or second-generation antipsychotics.Some antipsychotics, such as clozapine, fluperlapine and melperone were found to produce weak catalepsy in rodents, with minimal extrapyramidal symptoms and serum prolactin elevation in humans, compared to the earlier typical antipsychotic drugs, such as haloperidol. Meltzer and Matsubara explored the basis of these differences by testing the affinity of 38 antipsychotics for the rat striatal dopamine Deristics . These fAtypical antipsychotics, like to the typical antipsychotics, are efficacious in the treatment of positive symptoms of schizophrenia but display relatively lower propensity to cause extrapyramidal side effects. However, it was claimed that the label of “atypical” is not fully justified as they are different from first-generation antipsychotics only in their side effect profile  –19. In f2R, D3R and serotonin 5-HT1A receptors (5-HT1AR) as well as antagonism at serotonin 5-HT2AR and 5-HT2B receptors (5-HT2BRs) is represented by aripiprazole, cariprazine and brexpiprazole. Amongst these three partial agonist antipsychotics, aripiprazole and brexpiprazole display preferential binding affinity for dopamine D2R  family: D1 and D5 receptors (D1-receptor family) stimulate cAMP signaling pathway through a Gsα G-proteins, whereas D2-, D3- and D4-receptors (D2-receptor family) inhibit cAMP signaling through a Gi/oα G-proteins  is the highest in basal ganglia , accumbens nuclei, substantia nigra, amygdala and the frontal cortex. The cortex, substantia nigra, hypothalamus and the hippocampus express low level of dopamine D5 receptors (D5Rs). High levels of D2Rs are found in the basal ganglia, while cortical regions express low level of these receptors. D2Rs are the primary drug targets in schizophrenia, Parkinson’s disease, restless leg syndrome and neuroendocrine tumors. Highest expression of dopamine D3Rs are found mainly in the limbic system , with minor/low levels of expression in cortical regions. Dopamine D4 receptors (D4Rs) are found with relatively low level of expression in the amygdala, hippocampus, hypothalamus, cortex and, in the substantia nigra  and behavioral effects and side-effect profiles. However, their common property is that all display medium-to-high affinity for dopamine receptors of D2R-subtype  under in vitro conditions  and their degree of D3R selectivity are summarized in First and second-generation antipsychotics possess diverse structural, pharmacological  and the free plasma levels of antipsychotics were also demonstrated  helices embedded in the cell membrane connected by three extracellular (EL) and three intracellular (IL) loops (The DL) loops . The C-t2R and D3R exhibits a high level of general sequence identity which is increased in the transmembrane helices forming a highly conserved orthosteric binding site (OBS)  see . The mos ligands , 58. Mor ligands , 60. The D3R see . The SBSmbering; ] is leucentation .2R and D3R  stimulate cAMP signaling pathway through Gsα G-proteins whereas D2R, D3R, and D4R (D2R family) inhibit this pathway through Gi/oα G-proteins. There exists cAMP- independent pathways such as the recently recognized ß-arrestin pathway which is thought to be involved in several physiological functions and drugs’ effects  and D3R inhibit the enzyme adenylyl cyclase (AC) through Gi/oα subtype of G-protein leading to inhibition of cAMP-PKA-pDARPP32-PPI pathway. However, differences may exist in the coupling efficiency of the two receptors and AC (or its subtypes).Upon activation, both isoforms of D2R and D3R can activate ERK/MAPK signaling albeit with different mechanisms: D2Rs are coupled to and activate through α-subunit of Gi/o protein following agonist stimulation whereas, D3R functions through Go or Gß subunit depending on the signaling machinery of the given cell line. Both D2Rs and D3Rs are positively coupled to ß-arrestin-Akt-GSK3 pathway. GSK3ß is expressed in several brain regions and plays important role in neuronal development, neurovegetative and psychiatric diseases such as schizophrenia or bipolar disorder  for D2Rs and D3Rs . Similarly, high level of correlation was found between the affinity of antipsychotics for the rat D2R and D3Rs using [3H](+)-PHNO radioligand , the region is involved in schizophrenia pathology, and that significant correlation existed between the affinity of antipsychotics for D2Rs and D3Rs, it was thought that D3R affinity may play a role in the therapeutic efficacy of antipsychotics and led to propose development of selective D3R antagonists as novel antipsychotics , which form a lipophilic cavity. Hydrogen bond interaction with the serines in the 5.42 and 5.46 positions is typical for agonist binding state in D3R  (Non-selective ligands most probably bind to both the Dum ions) . These rum ions) .3R can be predicted at a reliable manner. In order to illustrate this, we docked several selected ligands into the D3R structures available in the Protein Data Bank  elicit catalepsy at higher doses  (3R-preferring D3R/D2R (such as S33138) antagonists  , 102 or agonists , 103 exeagonists .3R receptors in humans although their involvement is assumed in central nervous system (CNS) diseases such as schizophrenia, Parkinson’s disease, addiction, anxiety, and depression or in the clinical effects of antipsychotics  began soon after the discovery of D3R. Several antagonists fulfilling the selectivity requirements such as SB-277011A -4-propyl-9-hydroxynaphthoxazine . (+)-PHNO was originally described as an orally acting, potent dopamine receptor full agonist (3R (Ki: 0.16 nM) vs. D2R (Ki: 8.5 nM) -PHN 8.5 nM) .11C]-(+)-PHNO was synthesized by Wilson et al. -PHNO highly binds to regions rich in D3Rs. Using selective compounds such as the D3R antagonists SB-277011A, GSK598809 or the D2R antagonist SV-156, [11C]-PHNO proved to be useful for the separation of D3R and D2R binding signal and quantification of D3Rs in the brain, thus becoming an important tool for the investigation of the in vivo D3R occupancy by antipsychotics -PHNO, D3Rs are labeled in the rat cerebellum L9,10 and D2R in the striatum. This is based on the finding that the selective D3R antagonist, SB-277011 inhibited [3H](+)-PHNO binding in CB L9,10 membranes but not in the striatum whereas, the opposite profile was obtained with the D2R selective antagonist, SV-156  with significantly lower level or no occupancy in VP, ICj and substantia nigra (SN) (D3R rich regions). In contrast, in the in vitro autoradiography experiments all these antipsychotics inhibited [3H]-(+)-PHNO binding in the above regions except CB L9,10. It was concluded that under in vivo conditions the above-mentioned antipsychotics occupy dopamine D2R but not D3Rs despite their significant affinity for D3Rs in vitro  and cerebellar L9,10 region (D3R rich) to determine their in vivo D3R and D2R occupancy. The affinity data are given in Kiss et al. (in vivo binding/uptake of intravenously given [3H](+)-PHNO binding/uptake in the rat striatum and cerebellar L,910 regions. The results are summarized in 3R, full agonists pramipexole and PHNO potently inhibited [3H](+)-PHNO binding of CB L,910 membranes with marked preference toward CB L9,10 D3Rs. Cariprazine, didesmethyl-cariprazine (DDCAR), asenapine, raclopride and amisulpride, produced dose-dependent inhibition of [3H](+)-PHNO binding/uptake both in the striatal and CB L9,10 regions. Raclopride and asenapine, however demonstrated high striatal vs. cerebellar selectivity  produced inhibition of [3H](+)-PHNO binding/uptake in the striatum and little or modest level of inhibition in the CB L9,10.We extended this approach and compared the s et al. . We alsoectivity . The ant2R and serotonin 5-HT2R antagonist (in vitro for human D2R and D3Rs (Ki: 0.28 nM). Using the in vivo [3H](+)-PHNO method it caused dose-dependent, high occupancy of striatal D2R and D3R in the rat CB L9,10. In agreement with our data (see above) risperidone, olanzapine and aripiprazole demonstrated high occupancy only in the striatum and moderate or no occupancy was noted in the CB L9,10 region -PHNO PET. This study demonstrated that these antipsychotics caused high D2R occupancy in the D2R-rich dorsal striatum, using either [11C]raclopride or [11C](+)-PHNO. However, they failed to show binding signal in the D3R-rich globus pallidus using [11C](+)-PHNO (11C](+)-PHNO PET were reported by Mizrahi et al. demonstrating that in drug-naive, first episode schizophrenia patients, olanzapine and risperidone resulted in high occupancy in the D2R-rich regions but not in the globus pallidus where even “negative occupancy” was noted , in agreement with data obtained in rats, achieved significant D3R occupancy in healthy volunteers -PHNO . Similaras noted . On the lunteers .11C]raclopride -PHNO confirmed the D2R occupancy of aripiprazole however, minor levels of D3R occupancy was detected  (11C](+)-PHNO binding in brain regions with varying D2R and D3R expression. It showed significant occupancy of both D2R and D3R, albeit with approximately 3–6-fold selectivity for D3R   dose-dep for D3R , 143.2R preference   . Occupanse range .3Rs  demonstrated antipsychotic-like activity in animal experiments  compared with D2R 1 ; 1 ; . The in i values for D3Rs the picture is further complicated since like D2R, D3R may also exist in low and high affinity state. Sokoloff et al. did not find differences between affinity of dopamine for D3R in the absence or presence of Gpp(NH)p (24 vs. 27 nM)  , 80. How. 27 nM)  and Seem. 27 nM)  reportedeceptors . Affinitin vivo microdialysis in rodents, under physiological conditions the extracellular,  dopamine concentrations are in the low nanomolar range in various brain regions, including n. accumbens (∼1.5–4.5 nM), striatum (∼2–5 nM), hippocampus (∼1 nM) and in subnanomolar range in the prefrontal cortex (∼0.3–0.6 nM) (As determined by –0.6 nM) , 148–15411C]-(+)-PHNO PET Caravaggio et al. estimated that the Kd (dissociation constant) of dopamine is 22–24 nM and they reported that concentration of dopamine is between 8 and 9 nM in the ventral striatum, caudate and putamen and 2.8 nM in the globus pallidus  seem to be resistant to EEDQ-induced inactivation in vivo sites  concentration dependently reduced the vo sites .N- p-iso-thiocyanatophenethyl-spiperone) did not alkylate D3Rs receptors in n. accumbens and in the island of Calleja at doses that resulted in blockade of D2Rs receptors in caudate and n. accumbens. On the other hand, under in vitro conditions when slices from the above regions were incubated with EEDQ or NIPS, both inhibited dopamine D2Rs as well as D3Rs and inhibition at D3R sites were prevented by dopamine in nanomolar concentration range whereas only millimolar concentration of dopamine was able to protect D2Rs. The authors concluded that their results 3 receptors in vivo is prevented by its high affinity for even minor concentrations of endogenous dopamine“are consistent with the view that alkylation of D”  also did not significantly change extracellular dopamine concentration in the medial prefrontal cortex  elevated the extracellular dopamine concentrations in the rat prefrontal cortex, hippocampus, n. accumbens and in the striatum , 162. Iteceptors  which mal cortex , 161, whl cortex  blonansel cortex , and clol cortex  resultedl cortex , 163.2R antagonists can inhibit dopamine uptake via D2Rs  with D3R occupancy ED50 doses PHNO uptake/occupancy, We compared the effects of selected antipsychotics, D3R) occupancy ED50 doses, the agonists (+)-PHNO and (-)-pramipexole reduced the striatal dopamine turnover index by about 50%, whereas antipsychotics such as asenapine, haloperidol, olanzapine, risperidone, and ziprasidone and the D2R preferring antagonist SV-156 greatly enhanced (by about 3–4-fold) striatal dopamine turnover index -PHNO uptake) which were much lower than that of necessary for 50% occupancy of CB L9, 10 D3Rs, all antipsychotics  caused much less increase in dopamine turnover index -pramipexole and (+)-PHNO (which is probably due to the D2R agonist effects manifested under in vivo conditions). At pharmacological doses, neither cariprazine nor its one of the major metabolite, DD-CAR did not seem to alter significant alteration in dopamine metabolism in rat striatum.The results of dopamine turnover studies, in agreement with microdialysis results, indicate that D2R and D3R affinity and selectivity of some D3R selective antagonist, agonists, and antipsychotics along with their D3R occupancy determined in rats or in human.In in vitro affinity data one may expect compounds with low-or sub-nanomolar affinities for both receptor subtypes, would show D2R as well as D3R occupancy in vivo. However, the preclinical and human occupancy studies summarized above do not necessarily support such a correlation.Based solely on the 3R-preferring agonist, (+)-PHNO and pramipexole as well as the antagonists  all display low- or sub-nanomolar D3R affinity and high selectivity for D3Rs in vitro. These compounds produced D3R occupancy in rat or human studies. The same  is applicable for the partial agonists, cariprazine and its metabolite, DD-CAR. Although aripiprazole and brexpiprazole displayed low nanomolar in vitro D3R affinity, their D3R selectivity was below 1, which could explain their lack of D3R occupancy in vivo. Among the currently used antipsychotics, only the D2R/D3R antagonist blonanserin, which has low- or sub-nanomolar affinity for these receptors has been shown to have significant in vivo occupancy for both receptors in rats. Second generation antipsychotics  with low D3R affinity (Ki: >3–10 nM) and selectivity resulted in negligible D3R occupancy.Both D3Rs in the rat or human brain comes from the use of [3H](+)-PHNO or the [11C](+)-PHNO radiotracers. Their use represented a great advance in the in vivo imaging of D3Rs and determination of occupancy of brain D3Rs by antipsychotics.Our knowledge about the occupancy of D3H](+)-PHNO or the [11C](+)-PHNO however, are not ideal ligands/tracers for several reasons. They may not be sensitive enough for more detailed mapping of D3Rs in regions having low D3R expression e.g., cerebral cortex. Although both display higher affinity than dopamine for D3R, they are still sensitive to endogenous dopamine  continues  may greatly change the affinity of the two receptors toward the agonist tracer and the affinity of drugs to be examined and their occupancy. Thus, the quest for better ligands (agonist or antagonist?) for the demonstration of brain Dontinues , 181.2Rs  there is no reliable information on the optimal level of D3R occupancy for manifestation of therapeutic effect.Moreover, in contrast with the known therapeutically optimal occupancy of antipsychotics at D2R and D3Rs in vitro. In agreement with the in vitro D2R affinity they show significant D2R occupancy in the rat and human brain at their antipsychotic-effective doses. However, as revealed by animal and human occupancy studies, despite the considerable in vitro D3R affinity, not all antipsychotics demonstrated brain D3R occupancy in vivo.All currently used antipsychotics display high-to-medium affinity for both DThere may exist several possibilities for this dichotomy, as outlined in the following:3Rs than for D2Rs and thus endogenous dopamine might, at least partly, keeps D3Rs occupied even under basal conditions.First, dopamine displays much higher affinity for D2Rs, leads to several-fold increase of extracellular dopamine. Further, Amato et al. demonstrated that antipsychotics initially suppress dopamine transporter (DAT) activity leading to increase of dopamine in synaptic cleft, a mechanism which represents a further possible alternative way to modulate extracellular dopamine -pramipexole and (+)-PHNO reduced dopamine turnover, i.e., they decrease extracellular dopamine and increase D3R availability. Administration of antipsychotics , due to antagonism of presynaptic and biosynthesis and release regulating Ddopamine . Thus, t3R antagonists (D3R vs. D2R selectivity ≥100) such as ABT-925, GSK595809 and SB-277011 (with the in vitro low nanomolar D3R) affinity produced high D3R occupancy in animal or human studies, indicating primary importance of selectivity to achieve D3R occupancy in vivo.Third, beside the effects on the endogenous dopamine levels, the D3R affinity and selectivity appear to be further factors of importance. All three selective D3R/D2R partial agonist cariprazine and the D2R/D3R antagonist blonanserin shows that, in the presence of relatively high affinity for D2Rs, subnanomolar affinity for D3Rs appears to be necessary for D3R occupancy in vivo. Further, cariprazine and the F17464 (subnanomolar affinity for D3R with 75-fold D3R vs. D2R), do not increase extracellular dopamine and hence are able to compete for D3Rs vs. extracellular dopamine.Example of antipsychotics such as the D2R/D3R partial agonist antipsychotics, aripiprazole and brexpiprazole is somewhat controversial. Both demonstrated low nanomolar affinity for D2Rs and D3Rs (with D2R preference) in vitro, with negligible effects on extracellular dopamine in vivo. However, both produced no or very low occupancy of D3Rs for which the likely explanation is the D2R preference.The case of Din vitro D2R/D3R affinity vs. their brain D3R occupancy in vivo, indicate that levels of extracellular dopamine (or its change) in different brain regions is a key factor regarding D3R occupancy. On the other hand, the compounds’ high  D3R affinity and/or high D3R vs. D2R selectivity are also important determining factors to achieve significant D3R occupancy in the brain.In conclusion, data reviewed and discussed here regarding the current antipsychotics’ BKi and BKr drafted the manuscript with several inputs from IL. All authors were participating in the final editing and critical revision of the article and approved the final version to be published.BKi, BKr, and IL were employees of Gedeon Richter Plc.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Packaging of the eukaryotic genome with histone and other proteins forms a chromatin structure that regulates the outcome of all DNA mediated processes. The cellular pathways that ensure genomic stability detect and repair DNA damage through mechanisms that are critically dependent upon chromatin structures established by histones and, particularly upon transient histone post-translational modifications. Though subjected to a range of modifications, histone methylation is especially crucial for DNA damage repair, as the methylated histones often form platforms for subsequent repair protein binding at damaged sites. In this review, we highlight and discuss how histone methylation impacts the maintenance of genome integrity through effects related to DNA repair and repair pathway choice.  The packaging of eukaryotic DNA with histone proteins forms the fundamental unit of chromatin called the nucleosome. Nucleosomes contains 146 base pairs of DNA wrapped around a histone octamer containing two each of the H2A, H2B, H3, and H4 histones . TherefoHistone proteins undergo various post-translational modifications (PTMs), such as phosphorylation, acetylation, methylation, ubiquitylation, and sumoylation ,2. TheseHistone H4 lysine 20 methylation is the only reported methylation site on H4 that has a role in maintaining genome integrity upon DNA damage . H4K20 mSET8-dependent monomethylation of histone H4K20 has also been shown to play an important role in chromatin organization upon cell exit from mitosis ,46,47,48Methylation of histone H3 at lysine 4 by Set1p histone methyltransferase is associated with transcriptional activation and a proper response to DNA damage. Ubiquitination of H2BK123 by Rad6/Bre1 is a prerequisite for methylation of H3K4 ,53,54. ISaccharomyces cerevisiae is responsible for all forms of H3K36 methylation  [The methylation of histone H3 at lysine 36 (H3K36) is catalyzed by the Set2 histone methyltransferase. Set2 in nd tri-) ,71. In hnd tri-) ,72. Methnd tri-) . H3K36 mnd tri-) . In yeasnd tri-) ,75,76. Ind tri-) . Severalnd tri-) . Depletind tri-) ,79,80. Hnd tri-) . Upon DNnd tri-) . Depletind tri-) ,81,82. Ond tri-) ,84. IR-ind tri-) ,86. Dimend tri-) . Depletind tri-) ,87. AlthUnlike most histone methylations that occur on histone tails, H3K79 methylation occurs within the globular domain of histone H3 ,88. H3K7C. elegans has two H3K9-specific methyltransferases, MET-2 (SETDB1 homolog) and SET-25 (G9a/ SUV39H1 related). MET-2 is responsible for H3K9me1 and H3K9me2, while SET-25 catalyzes the final trimethylation step of H3K9 [Histone H3 methylation at lysine 9 (H3K9me3) is mainly associated with heterochromatin-mediated gene silencing ,101. H3K of H3K9 . MET-2-m of H3K9 ,108,109. of H3K9 . Silenci of H3K9 . Double  of H3K9 . H3K9 me of H3K9 . The int of H3K9 ,104. Met of H3K9 . ATM act of H3K9 . Acetyla of H3K9 ,42. Thus of H3K9 . Histone of H3K9 ,112,113. of H3K9 . Cells d of H3K9 ,111.As part of the cellular response to DNA damage, a wide range of histone PTMs  have been shown to play important roles in generating and regulating DDRs. In this review, we discussed the role of specific histone methylation sites ad enzymes in DNA DSB repair, and how they regulate pathway choice. Many histone methyltransferases and demethylases are recruited to chromatin in response to damage and change the local chromatin structure to facilitate repair-protein recruitment. However, there are still several gaps that need to be addressed to fully understand the role of histone methylation in DNA repair. Several histone methyltransferases and demethyltransferases target the same histone site, how is the activity of these enzymes regulated or their targeting to the same genomic loci? Sometimes the same modification can regulate both HR and NHEJ depending on the level of modification, so knowing the whole repertoire of methyl readers would shed light on how these modifications regulate different repair pathways. It has also been shown that both histone methyltransferases and demethylases against specific methylations play a role in DNA repair, but how the activities of these two opposing enzymes regulate the same outcome in the context of DNA repair needs to be elucidated further. Mutations of various methylation sites, or misregulation of methyltransferases or demethylases, and the related failure to repair damaged DNA in various diseases need to be further analyzed. The nature of the chromatin state before and after DNA damage, how that structure varies between different genomic loci, and the influence of specific types of DNA damage on repair outcomes are all challenging questions that need to be addressed.


In May, 2017, an outbreak of rotavirus gastroenteritis was reported that predominantly impacted Aboriginal children ≤4 years of age in the Kimberley region of Western Australia. G2P[4] was identified as the dominant genotype circulating during this period and polyacrylamide gel electrophoresis revealed the majority of samples exhibited a conserved electropherotype. Full genome sequencing was performed on representative samples that exhibited the archetypal DS-1-like genome constellation: G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2 and phylogenetic analysis revealed all genes of the outbreak samples were closely related to contemporary Japanese G2P[4] samples. The outbreak samples consistently fell within conserved sub-clades comprised of Hungarian and Australian G2P[4] samples from 2010. The 2017 outbreak variant was not closely related to G2P[4] variants associated with prior outbreaks in Aboriginal communities in the Northern Territory. When compared to the G2 component of the RotaTeq vaccine, the outbreak variant exhibited mutations in known antigenic regions; however, these mutations are frequently observed in contemporary G2P[4] strains. Despite the level of vaccine coverage achieved in Australia, outbreaks continue to occur in vaccinated populations, which pose challenges to regional areas and remote communities. Continued surveillance and characterisation of emerging variants are imperative to ensure the ongoing success of the rotavirus vaccination program in Australia.  Wide93, Melbo01 ,17. WThe aim of this study was to describe the epidemiology and burden of disease during an outbreak of rotavirus in the metropolitan (METRO) region of Perth and the remote Kimberley (KIMB) region of WA in 2017. Whole genome sequencing was performed to characterise the rotavirus strain circulating during this outbreak and place it in the context of global strains.In 2017, there were 519 notified cases of rotavirus infection in WA , making rotavirus the third most commonly notified enteric infection in WA. A marked increase in rotavirus notifications was noted in the second quarter of 2017 , with 236 cases, compared to the five-year second quarter average (2012–2016) of 100.8 cases .n = 122), compared to 52 cases in April and 62 cases in June. Of the 122 cases in May, 80 were aged ≤4 years (n = 26) and 1 year (n = 31). While cases were seen in all PHUs, Aboriginal people from the KIMB (n = 46) region and non-Aboriginal people from the metropolitan (METRO) region (n = 36) were the two most affected groups . Of these, 41% were fully vaccinated (48/118), 24% were partially vaccinated (29/118), and 35% were not vaccinated (41/118) . Only fiHospitalisation status was known for 78% of cases (95/122). For those with known hospitalisation status, 38% (36/95) were hospitalised as a result of their infection, of which 64% were Aboriginal people (23/36) and 36% were non-Aboriginal people (13/36). Children aged ≤4 years represented 86% of hospitalisations (31/36), of which Aboriginal children accounted for 71% (22/31). Of the hospitalised cases, 39% were fully vaccinated (14/36) and 36% were partially vaccinated (13/36). A further 22% were not vaccinated (8/36), five of which were ineligible for vaccination due to age. Vaccination status was unknown for one case.Cases were designated to the month of May based on the optimal date of onset (ODOO). Of the 122 cases from the month of May, a stool sample was available for 70 and were sent for genotype analysis at the National Rotavirus Reference Centre (NRRC), Murdoch Children’s Research Institute in Melbourne, Australia. The predominant genotype identified was G2P[4]  , with thRotavirus vaccination information was available for 94% (66/70) of cases notified in May with genotyping results . Of thesHospitalisation status was known for 86% of the genotyped cases in May (60/70), with 19 cases hospitalised. Of these, 47% were fully vaccinated (9/19), 26% were partially vaccinated (5/19), 16% were not vaccinated (3/19), and vaccination status was unknown for two cases (10%).A total of 38 G2P[4] samples were analysed using polyacrylamide gel electrophoresis to visualise the electropherotype pattern. The majority of samples had a highly similar electropherotype, indicating that a relatively conserved strain was circulating during the outbreak (data not shown).Three samples were selected for whole genome sequencing, which were representative of the dominant electropherotype: RVA/Human-wt/AUS/WAPC2769/2017/G2P[4] , RVA/Human-wt/AUS/WAPC2784/2017/G2P[4] , and RVA/Human-wt/AUS/WAPC2824/2017/G2P[4] . The three samples exhibited the archetypal DS-1-like genome constellation: G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2.The 11 genes of each sample were successfully sequenced, with the exception of the VP2 gene of RVA/Human-wt/AUS/WAPC2769/2017/G2P[4] and RVA/Human-wt/AUS/WAPC2824/2017/G2P[4] for which only 69.7–70.3% of the open reading frame (ORF) could be determined. The sample volumes were exhausted, attempting to resolve the approximate 800-base pair (bp) region at the 3′ prime end of the gene without success.The coding regions of each gene of RVA/Human-wt/AUS/WAPC2784/2017/G2P[4] and RVA/Human-wt/AUS/WAPC2824/2017/G2P[4] were highly conserved, with RVA/Human-wt/AUS/WAPC2769/2017/G2P[4] displaying some minor variability: VP1 (99.81–99.94% nucleotide (nt) and 99.82–99.91% amino acid (aa) similarity), VP2 (99.84–100% nt and 99.84–100% aa similarity), VP3 (99.84–99.92% nt and 99.88–100% aa similarity), VP4 (99.79–99.91% nt and 99.61–99.87% aa similarity), VP6 (99.92–100% nt and 100% aa similarity), VP7 (99.89–100% nt 99.69–100% aa similarity), NSP1 (99.86–99.93% nt and 100% aa similarity), NSP2 (99.90–100% nt and 100% aa similarity), NSP3 (99.47–99.79% nt and 99.39–99.68% aa similarity), NSP4 (99.62–99.81% nt and 99.43–100% aa similarity), and NSP5/6 (99.86–99.93% nt and 100% aa similarity).Phylogenetic analysis of the 11 genome segments was conducted to investigate the genetic relationships of the three outbreak samples RVA/Human-wt/AUS/WAPC2769/2017/G2P[4], RVA/Human-wt/AUS/WAPC2784/2017/G2P[4], and RVA/Human-wt/AUS/WAPC2824/2017/G2P[4] to previously characterised Australian samples and global strains a–k. In tAcross the VP1, VP2, VP3, and VP6 gene trees, the outbreak samples from this study continued to form conserved clusters with the contemporary Japanese samples RVA/Human-wt/JPN/MI1132/2016/G2P[4], RVA/Human-wt/JPN/K-21-16/2016/G2P[4], RVA/Human-wt/JPN/K-3-16/2016/G2P[4], RVA/Human-wt/JPN/Tokyo17-16/2017/G2P[4], and RVA/Human-wt/JPN/CH1020/2016/G2P[4] as observed in the VP7 and VP4 trees c–f. The Across the NSP1, NSP2, NSP3, NSP4, and NSP5 gene trees, the 2017 outbreak samples exhibited the same pattern across all trees: clustering with same group of contemporary Japanese samples, and falling within conserved clades comprised of G2P[4] strains from Belgium and Hungary that were detected in 2012, and Australian 2010 samples g–k.The 2017 outbreak samples were not closely related to the samples RVA/Human-wt/AUS/V233/1999/G2P[4], RVA/Human-wt/AUS/336190/2004/G2P[4] and RVA/Human-wt/AUS/V203/2009/G2P[4], which were associated with prior outbreaks in the Northern Territory, often clustering in separate lineages or distinct clades. This suggests that the current outbreak variant was not derived from the prior G2P[4] outbreak variants that had undergone genetic drift or reassortment over the intervening years but were more closely related to a G2P[4] variant that has been detected in Japan, Hungary, and other regions of the world. It may be derived from the Australian 2010 G2P[4] variant that has undergone moderate genetic drift during global circulation.The VP7 gene of the 2017 outbreak samples possessed 93.37–99.48% nt and 94.79–95.01% aa similarity with the G2 VP7 gene of RotaTeq. The amino acid differences between the outbreak samples and RotaTeq were analysed and 16 residues differed between the G2 component of RotaTeq and the two outbreak samples RVA/Human-wt/AUS/WAPC2824/2017/G2P[4] and RVA/Human-wt/AUS/WAPC2769/2017/G2P[4]. RVA/Human-wt/AUS/WAPC2784/2017/G2P[4] had 17 residues that differed. The altered residues that fell between amino acid 78 and 312 were mapped to the surface of the VP7 monomer to highlight mutations in proximity to the VP7 antigenic epitopes 7-1a, 7-1b, and 7-2  Figure . MutatioRotavirus was gazetted as a notifiable disease in WA in 2006 in part to monitor the effectiveness of the rotavirus vaccine when it was added to the childhood immunisation schedule in Australia in mid-2007 . The intA total of 236 rotavirus notifications were recorded in the second quarter of 2017, compared to the five-year second quarter average of 100.8 notifications, highlighting the scale of the outbreak. The five-year second quarter average was somewhat skewed by an outbreak in the second quarter of 2015  that affIn contrast to 2015, the increase in the second quarter of 2017 was noted to disproportionally affect young Aboriginal children in the KIMB region, which is in the north of the state. A number of towns and Aboriginal communities in the KIMB region were affected. The KIMB PHU investigated the increase in notifications, with assistance from local government environmental health officers. Several public health interventions were implemented as a result of their investigations, including the distribution of a public health alert to local hospitals and Aboriginal medical service providers, liaising with environmental health officers and community health staff to provide public health advice for affected communities, and an interview on local radio.It is noteworthy that an increased burden of rotavirus disease was reported elsewhere in Australia for 2017. Multiple outbreaks were recorded across Australia, due to equine-like G3P[8] in New South Wales and G8P[8] in New South Wales and Victoria . A compaThe inclusion of rotavirus vaccines into the Australian NIP in 2007 has resulted in a considerable and sustained decrease in rotavirus morbidity across most of Australia, with a 71% decline in rotavirus-coded hospitalisations of children aged <5 years reported . HoweverWhen the vaccination status of cases from the May peak was compared to hospitalisation status, vaccination did not appear to impact on whether a case was hospitalised. Fully or partially vaccinated children represented 75% of hospitalised cases 27/36) compared to unvaccinated eligible children accounting for 8% of hospitalised cases (3/36). Vaccination status was unknown for 3% of cases (1/36) and the remaining 14% of cases (5/36) were ineligible to have been vaccinated based on age. In May 2017, RotaTeq was the vaccine prescribed in the WA vaccination schedule and the vast majority of cases who were either fully or partially vaccinated were vaccinated with RotaTeq. Whilst a genotype-specific vaccine effectiveness has not been estimated for children in WA, the vaccine effectiveness of three doses of RotaTeq has been estimated at 82% (95% CI: 59–92) [ comparedFull genome sequencing was performed on representative samples from the outbreak. These samples were found to be most closely related to Japanese G2P[4] strains detected in 2016 and 2017 across all genes in the genome. In one associated paper, these closely related samples were reported as minor G2P[4] variants circulating in the Mie prefecture in 2017 . HoweverThe VP7 gene of the 2017 WA outbreak samples was compared to the G2 VP7 gene component of the RotaTeq vaccine. A total of 16 residues differed between the G2 component of RotaTeq and the two outbreak samples RVA/Human-wt/AUS/WAPC2824/2017/G2P[4] and RVA/Human-wt/AUS/WAPC2769/2017/G2P[4], and RVA/Human-wt/AUS/WAPC2784/2017/G2P[4] had 17 residues that differed. However, this is not unexpected as the RotaTeq G2 VP7 gene is derived from a strain that was circulating in 1992; global strains have undergone extensive genetic drift over the intervening years. Three of these altered residues in all three outbreak samples were observed in antigenic epitopes at positions A87T and D96N in antigenic region 7-1a, and S213D in region 7-1b . AlteredA limitation of this study was that a stool sample was available for 70/122 cases from the May peak. Not genotyping all samples could result in the proportion of the different genotypes being over- or underestimated. However, it does not alter the result that G2P[4] was the dominant genotype in the KIMB region as 41/53 samples were available and genotyped. The 70 samples available for genotyping were representative of the age distribution of rotavirus cases in WA during this period. However, more samples from Aboriginal cases were genotyped compared to non-Aboriginal cases (76% vs. 45%) and this could have overestimated the proportion of G2P[4] cases reported. Similarly, more samples were genotyped from the remote areas of the KIMB and PILB regions, which may also have overestimated the proportion of G2P[4] cases seen. Given this study largely focuses on the KIMB region, it is unlikely that this had any major impact on the overall results of the study.® Excel 365 . The ODOO is a composite of the ‘true’ date of onset provided by the notifying doctor or obtained during case follow-up, the date of specimen collection for laboratory notified cases, and when neither of these dates is available, the date of notification by the doctor or laboratory, or the date of receipt of notification, whichever is earliest. Notification data are broken down by regions that are based on Public Health Unit (PHU) boundaries, reflecting WA Health administrative regions: Central/Wheatbelt (CENT), Goldfields (GOLD), Great Southern (GSTH), Kimberley (KIMB), Metropolitan Perth (METRO), Midwest (MIDW), Pilbara (PILB), and South West (STHW).Data on WA cases of rotavirus were obtained from the WA Notifiable Infectious Disease Database (WANIDD). The notifications contained in WANIDD are received from medical practitioners and pathology laboratories under the provisions of the Public Health Act 2016 and subsequent amendments, and are retained in WANIDD if national case definitions are met. Rotavirus was listed as a notifiable disease in WA in July 2006 . Data waRecords of vaccine administration were submitted to the Australian Immunisation Register (AIR) . The AIR includes vaccines administered under the national immunisation program, school programs, and privately. CDCD staff accessed AIR to determine the rotavirus vaccine status of notified cases.A total of 122 faecal samples collected from children and adults presenting to hospital or general practice clinics with severe gastroenteritis in Western Australia during May, 2017 were determined to be rotavirus positive by a local diagnostic laboratory. Seventy de-identified rotavirus positive specimens were sent to the National Rotavirus Reference Centre (NRRC) at the Murdoch Children’s Research Institute. A further 27 samples did not have adequate remaining volume and were not sent for genotyping. There is no agreement with private pathology laboratories to forward samples for genotyping. As a result, 25/122 (20%) of samples were not genotyped. Where possible, metadata, including date of collection, date of birth, gender, and postcode, were collected. Samples were stored at −80 °C until analysis, allocated a unique laboratory code, and entered into a REDCap database.w/v) faecal extracts using the QIAamp Viral RNA mini extraction kit  according to the manufacturer’s instructions. Rotavirus G- and P-genotyping was performed using a hemi-nested multiplex RT-PCR assay  outbreak disproportionately impacted Aboriginal children ≤4 years of age in the remote Kimberley region of Western Australia. The G2P[4] variant circulating was closely related to contemporary Japanese G2P[4] samples, suggesting a global variant that exhibited the altered residues A87T, D96N, and S213D compared to the G2 component of the RotaTeq vaccine, residues that have been observed in the majority of G2P[4] strains circulating globally over the last two decades. Despite national vaccine coverage of 85.4%, outbreaks continue to occur in vaccinated populations in Australia, in particular impacting Aboriginal populations. These outbreaks pose particular challenges to regional areas and remote communities. Continued surveillance and characterisation of emerging variants are imperative to ensure the ongoing success of the rotavirus vaccination program in Australia.


Hepatitis B virus (HBV) expresses co-terminal large (L), middle (M), and small (S) envelope proteins. S protein drives virion and subviral particle secretion, whereas L protein inhibits subviral particle secretion but coordinates virion morphogenesis. We previously found that preventing S protein expression from a subgenomic construct eliminated M protein. The present study further examined impact of S protein on L and M proteins. Mutations were introduced to subgenomic construct of genotype A or 1.1 mer replication construct of genotype A or D, and viral proteins were analyzed from transfected Huh7 cells. Mutating S gene ATG to prevent expression of full-length S protein eliminated M protein, reduced intracellular level of L protein despite its blocked secretion, and generated a truncated S protein through translation initiation from a downstream ATG. Truncated S protein was secretion deficient and could inhibit secretion of L, M, S proteins from wild-type constructs. Providing full-length S protein in trans rescued L protein secretion and increased its intracellular level from mutants of lost S gene ATG. Lost core protein expression reduced all the three envelope proteins. In conclusion, full-length S protein could sustain intracellular and extracellular L and M proteins, while truncated S protein could block subviral particle secretion. Hepatitis B virus (HBV) is an enveloped DNA virus with a small circular genome of 3.2 kb ,2. The 1HBV genome replication initiates with the assembly of core protein into core particles, packaging pregenomic RNA (pgRNA). pgRNA is converted by the co-packaged DNA polymerase (P protein or pol) into partially double stranded DNA. L protein initiates virion morphogenesis by interacting with core particles through its matrix domain ,7. L proA subgenomic (0.7 mer) construct of the HBV genome inserted to a cloning vector could express all the three envelope proteins under endogenous SPI and SPII promoters A. We preATGTGTCTG to CGGGATGGGTCTG  or CAGGATGGGTCTG , and diminished by its mutation to TTTTATGTGTCTT . Many mutations in the S region caused amino acid substitutions in L and M proteins, and sometimes in S protein if it was expressed (Construct N55 had the S gene (nt155-835) of HBV6.2 of genotype A cloned to pcDNA3.1/Zeo(-) vector  to allow S protein expression under the CMV promoter A. The 0.xpressed .- mutant had C48* nonsense mutation (C2044A in geno5.4 and T2044A in geno1.2 at the nucleotide level) (- mutant had W52* (G3008A) nonsense mutation for geno5.4 but K38* (A2959T) nonsense mutation for geno1.2, with the latter accompanied by Q218L substitution in P protein. The S- mutant of all the three clones had the S gene start codon mutated to ACG. Two preS2 mutations were introduced to the infectious genotype D clone [The 1.1 mer construct was generated by inserting nt1805-3221/1-1932 of a genotype A clone or nt1805-3182/1-1932 of two genotype D clones into the SacI—HindIII sites of pcDNA3.1 zeo(-) vector A. The gee level) . The L- The transfection experiments and Western blot analysis are well established ,22,23,24HBsAg and preS1 antigens in culture supernatant and cell lysate were measured by enzyme linked immunosorbent assay (ELISA) using commercial kits  according to the manufacturer’s direction. Different dilutions were used for samples with high vs. low HBsAg and preS1 antigen titers to avoid signal saturation.2 and 12 mM MgCl2. Core particles were precipitated overnight by adding 38 μL of PEG solution , resuspended in 50 μL of 10 mM Tris (pH7.5)/8 mM CaCl2/6 mM MgCl2, and further treated with 3U of mung bean nuclease and 2U of DNase I at 37 °C for 30 min. Core particles were disrupted by addition of 150 μL of proteinase K digestion buffer , and digested overnight at 55 °C with 0.5 mg/mL of proteinase K . DNA was extracted with Tris-saturated phenol (Sangon Biotech), and precipitated with ethanol. Purified DNA was dissolved in Tris buffer and separated in 1.3% agarose gel. Following overnight transfer to a nylon membrane (Roche), the blot was hybridized with α-32P-labeled probe of genotype D (purified 3.2-kb HBV DNA devoid of vector sequence). The signals on the phosphor screen were scanned by Typhoon FLA 9500 software. Virions were immunoprecipitated from 1.5 mL of culture supernatant by overnight incubation at 4 °C with rabbit anti-preS1 antibody (Genscript) and rabbit anti-S antibody (Novus) preconjugated to protein G-Sepharose beads. The precipitate was washed with PBS buffer twice and resuspended in 50 μL of 10 mM Tris (pH 7.5)/8 mM CaCl2/6 mM MgCl2. Samples were treated with 3U of mung bean nuclease and 2U of DNase I at 37 °C for 30 min. This was followed by addition of 150 μL of the proteinase K buffer, and digestion with 0.5 mg/mL of proteinase K at 55 °C overnight. DNA was extracted with Tris-saturated phenol and precipitated with ethanol using 1 μL salmon sperm DNA and 1 μL glycogen (Roche) as carriers. Purified virion DNA was subject to Southern blot analysis just as for intracellular replicative DNA.A total of 2 μg plasmid DNA was transfected into Huh7 cells seeded in 6-well plates. Cells and culture supernatant were harvested four days later. Details of HBV DNA analysis have been described ,21,22,24t-test. * p < 0.05 is considered as statistically significant.All experiments were repeated for three times, and the data were presented as mean with standard deviation (SD). Statistical analysis was performed by GraphPad Prism 6 software using a Student’s +/M+/S+), a 0.7 mer construct of a genotype A clone capable of efficient expression of the three envelope proteins under endogenous SPI and SPII promoters [We previously generated N51  to provide full-length S protein in trans. Alternatively they were co-transfected with empty vector (pcDNA3.1) to serve as a negative control. N65 was derived from N51 (L+/M+/S+) by a nonsense mutation of preS2 codon 43 (S43*). N65 was also co-transfected with pcDNA3.1 or N51. Its co-transfection with N51 moderately increased intracellular levels of L and M proteins  substitutions in L protein: M175A, M175I, M175K, and M175L . To valiAlthough data presented in +/M+/S+) or 1.1 mer replication construct of genotype A (geno5.4) or genotype D (geno1.2) was co-transfected with increasing amount of N65-tS+ overexpressing gp19/p16, with the total amount of co-transfected plasmid DNA kept constant using N65-nS (incapable of expressing any envelope protein). Although the intracellular level of gp19/p16 increased according to the amount of N65-tS+ co-transfected, the intracellular level of L protein and full-length S protein did not show a clear dose-dependent increase  and a genotype D clone (geno1.2) by the C48* nonsense mutation . Blotting with the anti-preS2 antibody confirmed such a finding  lost intracellular M protein and had much reduced L protein . The S-/core- mutant displayed even lower intracellular level of L protein than either S- or core- mutant . While provocative, these findings are preliminary. We cannot exclude the possibility that lost core protein expression somehow reduced 2.4-kb and 2.1-kb RNAs for envelope proteins. It remains to be determined whether similar phenotypes can be reproduced by ablating core protein expression through a different mutation (such as lost ATG codon). If the core protein can indeed stabilize the three envelope proteins, then co-transfection of the 1.1 mer core- mutant with an expression construct for core protein should rescue intracellular and extracellular levels of L, M, and S proteins.The current study started to explore a possible role of core protein in sustaining the envelope proteins. We found that lost core protein expression via the C48* nonsense mutation diminished both intracellular and extracellular levels of L protein  could dose-dependently inhibit HBsAg secretion from the WT construct, whether as 0.7 mer construct or 1.1 mer replication construct C. Wester


Importantly, epitope mapping analysis has revealed that N1mAbs with growth inhibitory effect recognize immunoglobulin-like and FNIII domains of NLRR1, which also indicates the importance of FNIII domain in the function of NLRR1. Thus, the present study provides a new insight into the development of a cancer therapy by targeting NLRR1 as a modulator of proliferative signals on cellular membrane of tumor cells.Receptor tyrosine kinases (RTKs) receive different modulation before transmitting proliferative signals. We previously identified neuronal leucine-rich repeat 1 (NLRR1) as a positive regulator of EGF and IGF-1 signals in high-risk neuroblastoma cells. Here, we show that NLRR1 is up-regulated in various adult cancers and acts as a key regulator of tumor cell proliferation. In the extracellular domains of NLRR1, fibronectin type III (FNIII) domain is responsible for its function to promote cell proliferation. We generated monoclonal antibodies against the extracellular domains of NLRR1 (N1mAb) and screened the positive N1mAbs for growth inhibitory effect. The treatment of N1mAbs reduces tumor cell proliferation  MYCN oncogene (Neuroblastoma (NB), originally arising from the sympathoadrenal lineage of the neural crest, is one of the most common extracranial solid tumors in childhood. NBs in patients less than 1 year of age often regress spontaneously, resulting in a favorable prognosis  functionally regulated by different mechanisms including gene expression, endocytosis, dephosphorylation, and crosstalk with other membrane proteins –9. Our pIn high-risk NB, despite a great improvement of its combinatorial therapy with surgical resection, intensive chemotherapy, radiotherapy, and immunotherapy, only 40 to 50% of patients survive long term , 16. TheIn the present study, we found the up-regulation of NLRR1 expression in various adult cancers and non-NB cell lines. Hybridomas producing monoclonal antibodies (mAbs) to extracellular part of NLRR1 were developed and subjected to screening assays. Monoclonal antibody against NLRR1 (N1mAb) with growth inhibitory effect was found to target the domains of NLRR1 responsible for its function to regulate cell proliferation. Furthermore, the treatment of N1mAb suppressed EGF signals, potentiated the effect of EGFR inhibitor, and decreased the tumor growth in mouse xenograft models.β-III tubulin (Tuj1) antibody (#MMS-435P) was from Covance ; anti-HA tag antibody (#11867423001) was from Roche; anti-BrdU antibody (#M0744) was from DakoCytomation ; anti-actin (#A5060) was from Sigma; and the sheep polyclonal anti-NLRR1 antibody (#AF4990) was from R&D Systems . AG1478 was from Calbiochem . Lung and prostate tissue lysate arrays (Tissue Lysate Dipstick Array) were from Protein Biotechnologies .EGF and IGF-I were from Sigma . Complete protease inhibitor cocktail and phosphatase inhibitor cocktail were from Roche ; 5-bromo-2-deoxyuridine (BrdU) was from Sigma; 3,3′-Dithiobis(sulfosuccinimidylpropionate) (DTSSP) was from Thermo Fisher Scientific . Antibodies against phospho-EGFR (#2236), phospho-ERK (#9101), phospho-Akt (#9271), EGFR (#4267), ERK (#9102), Akt (#9272), and myc tag (#2276) were from Cell Signaling Technology ; anti-NLRR1 (Assay ID: Hs00979743_m1) and β-actin control reagent kit were purchased from Applied Biosystems. The mRNA levels of each gene were standardized by β-actin. All human neuroblastoma specimens used in the present study were obtained at various institutions and hospitals in Japan and provided to the Chiba Cancer Center Neuroblastoma Tissue Bank with appropriate informed consent. The procedure of this study was reviewed and approved by the internal review board of Chiba Cancer Center.Total RNA was extracted from 12 lung adenocarcinoma or nine squamous cell carcinoma and adjacent non-cancerous tissues as well as NB and non-NB cell lines using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions, and reverse transcription was performed with SuperScript II reverse transcriptase (Invitrogen). qRT-PCR was carried out using 7500 Real-Time PCR System (Applied Biosystems), according to the manufacturer’s protocol. TaqMan probe for HEK293 and MCF7 cells were cultured in Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum . Human neuroblastoma SH-SY5Y and SK-N-BE(2) cells were maintained in RPMI 1640 (Sigma) supplemented with 10% FBS. HEK293 cells were obtained from the JCRB Cell Bank and MCF7 and SH-SY5Y cells were from ATCC, while SK-N-BE(2) cells were purchased from the European Collection of Authenticated Cell Cultures. Mycoplasma contamination was tested by Mycoplasma Detection Set (Takara), and short tandem repeat analysis was performed for cell authentication (Promega). Transient transfection with C-terminal hemagglutinin (HA) or myc-tagged human NLRR1 plasmids was performed using Fugene HD (Roche) or Lipofectamine2000 (Invitrogen) according to the manufacturer’s instructions. Seventy to eighty percent confluent monolayers of transfected cells were treated with growth factors for the indicated times after 16 h serum starvation, and cell lysates were subjected to western blot analyses. Cell proliferation was determined using a Cell Counting Kit-8 .SK-N-BE cells were infected with four different MISSION lentiviral particles encoding NLRR1 shRNAs (Sigma). NLRR1 shRNA target sequences were as follows: #1, CCGGCCACAACTTTGCGTATGTGAACTCGAGTTCACATACGCAAAGTTGTGGTTTTTTG; #2, CCGGCCACCTGAACTCCAACAAATTCTCGAGAATTTGTTGGAGTTCAGGTGGTTTTTTG; #3, CCGGGCTGAACAACAATGCCTTGAACTCGAGTTCAAGGCATTGTTGTTCAGCTTTTTTG; and #4, CCGGGCTAGACTTGTTACCTTCGTTCTCGAGAACGAAGGTAACAAGTCTAGCTTTTTTG. Stable cell lines were generated by selection with puromycin (0.8 µg/ml). To exclude the possibility of off-target effects of NLRR1 shRNAs, 3′UTR-targeted #4 shRNA stably expressing SK-N-BE cells were transiently introduced with pcDNA3-NLRR1-HA expression plasmid and subjected to cell proliferation assays, as above.Human tissue array slides were obtained from Super Bio Chips  and subjected to immunostaining using polyclonal anti-NLRR1 antibody (1:25). BrdU was injected intraperitoneally into mice bearing tumors at a dose of 100 μg/g body weight 24 h before autopsy. Tumor tissues were fixed in 4% paraformaldehyde, dehydrated with a graded ethanol series, and embedded in paraffin. Sections (4 µm) were deparaffinized by immersion in xylene and rehydrated, followed by immunostaining for BrdU. Number of BrdU-positive cells was counted in four high power fields (hpfs) of each tumor at ×400 magnification.HEK293 cells expressing HA-tagged and myc-tagged NLRR1 were treated with cross-linker (DTSSP) for 2 h at 4°C. The cell lysates (500–750 μg) were incubated with appropriate antibodies for 1 h at 4°C, followed by incubation with protein G-agarose at 4°C overnight. After extensive washing of the beads with lysis buffer, the immunoprecipitates were detected by western blot analyses. Total cell lysates were resolved by SDS-PAGE followed by western blot detection using the indicated antibodies .HEK293 cells were transiently transfected with C-terminal tagged human NLRR1, NLRR2, or NLRR3 plasmids and treated with EZ-Link Sulfo-NHS-LC-LC-Biotin (Thermo Fisher Scientific) or vehicle alone for 30 min at room temperature. Cell lysates (500 μg) were incubated with NeutrAvidin Agarose Resin (Thermo Fisher Scientific). The precipitated proteins were resolved by SDS-PAGE followed by western blot detection using appropriate antibodies.5/ml in the medium containing 50% of the conditioned medium from the hybridomas. For the positive hybridomas, the monoclonal antibodies were purified by protein A Sepharose column chromatography by MBL.The monoclonal antibodies were generated against extracellular domain of NLRR1 . The purified proteins of NLRR1 extracellular domain and complete Freund’s adjuvant (1:1) were injected into mice. Three days after the final injection, lymph-node cells were removed from immunized mice and were fused with P3U1 myeloma cells at a ratio of 5:1 by the polyethyleneglycol-400 procedure. Cultured supernatants of the hybridomas were screened in transfectants expressing NLRR1 by flow cytometric analysis (FCM) and the binding assay in 96-well plate coated with NLRR1 proteins. After cloning of the hybridomas which showed the positive results in FCM and/or the binding assay, the culture supernatants from wells were tested for growth inhibitory effect by culturing CHP134 cells at 1 × 10The isotype of each of the N1mAbs was determined by MBL. The plasmids for NLRR1 lacking the extracellular domains  were prepared using Infusion . Peptide microarray was generated by JPT Peptide Technologies GmbH  and analyzed by SureScan Microarray Scanner . For flow cytometry, HEK293 cells were transiently transfected with the plasmids of NLRR1 lacking the extracellular domains and resuspended in phosphate-buffered saline (PBS). The cells were incubated with N1mAbs for 1 h at 4°C followed by the incubation with Alexa Fluor 488-labeled anti-mouse IgG (Thermo Fisher Scientific). The cells were analyzed using FACSCalibur .6 SH-SY5Y cells stably expressing NLRR1 and CHP134 cells in 0.1 ml of PBS/Matrigel. After implantation, tumor sizes were measured using the following formula: [(width)2 × length]/2. After tumors became >75 mm3, mice were randomized into two groups and intraperitoneally administered with vehicle or NLRR1 monoclonal antibody #281 twice a week for 3 weeks. For in vivo imaging, N1mAb 281 was labeled with HiLyte Fluor 750 using AnaTag Protein Labeling Kit  and visualized using a Lumazone imaging system  after injection into the tail vein. All mice were maintained in a specific pathogen-free animal facility. All animal experiments were approved by the Animal Care and Use Committee of Chiba Cancer Center Research Institute.SH-SY5Y cells stably expressing NLRR1 were established by transfection followed by selection with G418 at concentration of 600 µg/ml for about 4–6 weeks . Seven-wt-tests (two-tailed) and ANOVA tests were employed to examine the differences between two groups and that of differences between more than two groups, respectively. In xenograft study, the difference between groups was evaluated by two-way repeated measures ANOVA followed by Bonferroni posttest. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 versus saline at each time; ##, P < 0.01 versus saline group.Student’s NLRR1 was also detected in NB cell lines and non-NB cell lines . High expression of NLRR1 was observed in tumorous tissues; eight out of twelve adenocarcinomas and three out of nine squamous cell carcinomas showed more than two-fold higher expression of NLRR1 than normal tissues , immunoglobulin-like (Ig), fibronectin type III (FNIII) domains and a short intracellular tail. To identify the functional domains of NLRR1, we constructed the expression vectors of NLRR1 with the deletions in extracellular domains . Cell growth assay in MCF7 cells expressing the deletion mutant NLRR1 revealed that the deletion of FNIII domain significantly reduced the cell growth compared to wild-type NLRR1  tumors. Even at relatively low dose , treatment of N1mAb led to significant suppression of tumor growth of SH-SY5Y/NLRR1 cells, strongly supporting the growth inhibitory effect of N1mAb (To elucidate the mechanism of growth inhibition by N1mAb, the binding property of N1mAb 281 was examined by immunoprecipitation and indirect flow cytometry. HEK293 cells overexpressing HA-tagged NLRR1 were subjected to immunoprecipitation assay. As shown in Next, we tested N1mAbs for the combinatorial effect with EGFR inhibitor because previous reports showed that EGFR is expressed in NB and adult cancers and could be a therapeutic target for these tumors , 22. TheTo understand the mechanism how N1mAb inhibits the function of NLRR1, we first performed immunoprecipitation assay using vectors for two different tagged NLRR1 and found that NLRR1 proteins form self-multimers S8A. BecWe demonstrate that NLRR1 expression is up-regulated in adult cancer tissues including lung and breast in addition to unfavorable NBs. NLRR1 is involved in determining the malignant status of cancer cells by enhancing the proliferative signaling of EGFR and IGF-IR. This is unique to NLRR1 among NLRR family members despite their close similarity in protein structures with high evolutional conservation . EGFR faTransmembrane proteins that function to regulate cell proliferation have been the most popular candidates for therapeutic targets to develop a novel remedy against cancers. In particular, kinase inhibitors and neutralizing antibodies have been developed against RTKs of which the knockdown expression shows a great repression of cell growth and survival. Given that NLRR1 knockdown repressed NB cell proliferation, these lines of evidence regarding NLRR1 function in the present study may provide us an attractive scientific basis for targeting NLRR1. Three possible mechanisms of the inhibitory effect by single usage of NLRR1 antibody are hypothesized: [1] inhibition of NLRR1 self-dimerization which is shown in Our present characterization of epitopes recognized by N1mAbs revealed that N1mAbs with growth inhibitory activity bind to Ig and FNIII domains of NLRR1 Figure 4We also tested combinatorial use of N1mAb with EGFR kinase inhibitor. At present, it has been proposed that partial inhibition of multiple targets could be more effective than full inhibition of a single molecule in NB therapy , 26. IndThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. The animal study was reviewed and approved by Animal Care and Use Committee of Chiba Cancer Center Research Institute.AT and AN conceived and designed the experiments. AT, SH, AO, JA, and YN performed the experiments. AT, SH, and AN analyzed the data. YN contributed reagents/materials/analysis tools. AT and AN wrote the paper. All authors contributed to the article and approved the submitted version.This work was supported by a Grant-in-Aid from the Japan Ministry of Health, Labour and Welfare for Third Term Comprehensive Control Research for Cancer to AN, JSPS KAKENHI Grant Number JP21390317, JP24249061 to AN, JP19890276 to AT, MEXT KAKENHI Grant Number JP22791016 to AT, and a grant from the Takeda Science Foundation to AN.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Human papillomavirus type 16 (HPV16) E1 and E6 proteins are produced from mRNAs with retained introns, but it has been unclear how these mRNAs are generated. Here, we report that hnRNP D act as a splicing inhibitor of HPV16 E1/E2- and E6/E7-mRNAs thereby generating intron-containing E1- and E6-mRNAs, respectively. N- and C-termini of hnRNP D contributed to HPV16 mRNA splicing control differently. HnRNP D interacted with the components of splicing machinery and with HPV16 RNA to exert its inhibitory function. As a result, the cytoplasmic levels of intron-retained HPV16 mRNAs were increased in the presence of hnRNP D. Association of hnRNP D with HPV16 mRNAs in the cytoplasm was observed, and this may correlate with unexpected inhibition of HPV16 E1- and E6-mRNA translation. Notably, hnRNP D40 interacted with HPV16 mRNAs in an HPV16-driven tonsillar cancer cell line and in HPV16-immortalized human keratinocytes. Furthermore, knockdown of hnRNP D in HPV16-driven cervical cancer cells enhanced production of the HPV16 E7 oncoprotein. Our results suggest that hnRNP D plays significant roles in the regulation of HPV gene expression and HPV-associated cancer development. Human papillomaviruses (HPV) contain a double-stranded circular DNA genome of approximately 8 kb in size . HPV infSince the HPV16 genome has two promoters only, alternative mRNA splicing plays a major role in the regulated expression of all HPV16 genes . A complHeterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large family of RNA-binding proteins (RBPs). The association of hnRNP proteins with pre-mRNAs is initiated co-transcriptionally at the nascent transcripts. Many of the RNA-binding proteins remain bound to the resulting mRNAs all the way to the ribosomes and shuttle back and forth between the nucleus and the cytoplasm, demonstrating that RNA-binding proteins are important determinants of pre-mRNA processing during the entire mRNA pathway including mRNA splicing, localization, translation and stability . hnRNPs The hnRNP family comprises at least 20 major RNA-binding proteins originally named alphabetically from A1 to U. These proteins share modular structures that include RNA recognition motifs (RRM) or quasi-RRMs, other motifs such as the K Homology (KH) domain and the arginine/glycine-rich RGG motif and other RNA-binding domains (RBD) present on a subset of hnRNPs . In someIn this manuscript, we show that hnRNP D proteins inhibit splicing and promote retention of the E1- and E6-encoding introns on the HPV16 early mRNAs, thereby specifically stimulating production of the partially spliced HPV16 E1- and E6-encoding mRNAs. Furthermore, hnRNP D facilitated export of the partially spliced HPV16 E1 and E6 mRNAs to the cytoplasm. However, despite the fact that the HPV16 E1 and E6 mRNAs reached the cytoplasm in association with hnRNP D, HPV16 mRNAs were poorly translated. In conclusion, our results suggest that hnRNP D proteins inhibit HPV16 early mRNA splicing and enhance production of the partially spliced, intron-containing HPV16 E1 and E6 mRNAs, thereby playing an important role at the initial steps of the biogenesis of HPV16 E1 and E6 mRNA.2 at a final concentration of 2.4 mM in the keratinocyte culture medium for 24 h. The HPV16-immortalized keratinocyte 3310 cell line has been described previously and was generated by stable transfection of nHFK with HPV16 genome plasmid pHPV16ANE2fs  (HyClone) with 10% bovine calf serum (HyClone) and penicillin/streptomycin (Gibco). C33A2 cell line has been described previously . Briefly16ANE2fs .The following plasmids have been described previously: pHPV16AN , pC97ELs′-end and E7 to a flag tag sequence at the 3′-end. pXH856F was generated by insertion of DNA fragment PCR-amplified with pXH856F sense and anti-sense primer  creating pXH856SDmF. The mutations in SD226 were introduced by PCR mutagenesis with pXH856SDmF sense and antisense primers (′-end and was constructed by PCR amplification using p16E1-3xF inverse sense and anti-sense primers (′-splice sites SD880 and SD1302 on p16E1SDm-3xF were mutated (G881C and G1303C) to create p16E1SDm-3xF. The mutations were conducted by p16E1SDm-3xF sense and antisense primers . Briefly, a mixture of Turbofect:DNA ratio of 2:1 (μl reagent: μg DNA) for HeLa cells or 4:1 for 293T cells and DMEM without serum was incubated at room temperature for 20 min prior to dropwise addition to subconfluent cells. Transfections of nHFK were performed with ViaFect according to the manufacturer’s protocol (Promega). In brief, a mixture of ViaFect:DNA ratio of 3:1 and EpiLife without serum was incubated at room temperature for 20 min prior to dropwise addition to subconfluent cells. Fluorescence images of EGFP set of plasmids transfected HeLa cells were acquired using Olympus CKX53 inverted microscope.Nuclear and cytoplasmic extracts were prepared from HeLa cells grown in 6 cm dishes at 24 h post-transfection. Cells were harvested by using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) according to the manufacturer’s protocol. In brief, cell pellets were resuspended in ice cold buffer CER I with protease inhibitors (Sigma Aldrich) and vortexed vigorously prior to incubation on ice for 10 min. Ice-cold buffer CER II was added to the samples that were vortexed vigorously and incubated on ice for one minute. After 5 min of maximum speed centrifugation, the supernatants were collected as cytoplasmic extracts. The pellets were washed once by PBS and collected as nuclear extracts.′-splice site SD226 to 3′-splice site SA409 was amplified with RT-PCR primers 97S and 438AS  and Direct-zol RNA MiniPrep (ZYMO Research) according to the manufacturer’s protocols. Reverse transcription (RT) was performed in a 20 μl reaction using random hexamers (Invitrogen) and reverse transcriptase (Invitrogen). One microliter of cDNA was subjected to PCR-amplification. cDNA representing HPV16 mRNAs spliced from HPV16 5nd 438AS  and cDNAnd 880AS . cDNA re or E4AS . cDNA reectively . Glyceraectively . Splicedectively . cDNA reectively . For locCt), and the relative expression levels were calculated using the 2∧ΔΔCt method. Results were normalized to GAPDH mRNA levels determined with primers GAPDHF and GAPDHR  using the SsoAdvanced SYBR Green Supermix (Bio-Rad) according to the manufacturer’s instructions. To quantitate intron retained E6 mRNAs by RT-qPCR primers TotalE6F and 234AS were used . For prid GAPDHR . mRNA quMetridia longa secreted luciferase (sLuc) activity in the cell culture medium of transfected cells was monitored with the help of the Ready-To-Glow Secreted Luciferase Reporter assay according to the instructions of the manufacturer (Clontech) as described previously (The eviously . In brieCell extracts for western blotting were obtained by resuspending transfected cells in radioimmunoprecipitation assay (RIPA) buffer consisting of 50 mM Tris HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, 1 mM DTT and protease inhibitor (Sigma Aldrich), followed by centrifugation at full speed for 20 min and collection of the supernatants. Proteins were denatured by boiling in Laemmli buffer. After SDS-PAGE, the proteins on the gels were transferred onto nitrocellulose membranes, blocked with 5% nonfat dry milk in PBS containing 0.1% Tween 20 and stained with specific primary antibodies  to the i2, 0.5% Triton X-100) in strips of PCR tubes incubated at RT for 20 min. To pull-down proteins, 15 μg HeLa whole cell lysate was mixed with beads bound with ssRNA oligos and incubated with rotation for 1 h at room temperature followed by washing of beads 10 times with binding buffer using DynaMag 96 Side magnetic plate (Invitrogen). Proteins were eluted by boiling of the beads in Laemmli Buffer. Samples were subjected to SDS-PAGE followed by western blot analysis with the indicated antibodies , Proteinase inhibitor (Sigma Aldrich) and 5% glycerol. Biotin-labeled ssRNA oligos were purchased from Sigma Aldrich (Supplementray Table S3). DynabeadsM-280 Streptavidin magnetic beads (Invitrogen) were bound to biotin-labeled ssRNA oligonucleotides in 200 μl of binding buffer  or IgG was added to Dynabeads protein G and incubated with cell lysates under gentle rocking at 4°C overnight. The complexes were washed six times using cell lysis buffer and eluted by boiling in Laemmli buffer. Samples were subjected to SDS-PAGE followed by western blotting with specific primary antibodies .−2 UV light (254 nm) in a bio-link cross-linker (Biometra). Cytoplasmic extracts were prepared as described above. Whole cell lysates were prepared by resuspending cells in one ml of RIPA buffer and incubated on ice for 30 min with occasional vortexing to lyse cells. For immunoprecipitations, 2μg of the anti-flag antibody  or mouse IgG was incubated at 4°C overnight in 0.5 ml of cell lysate. About 20 μl of Dynabeads Protein G  and 20 μl Dynabeads Protein A  were blocked with 1% BSA for 0.5 h, washed three times in RIPA buffer and then added to the antibody–protein mixtures followed by incubation for 1 h at 4°C. The beads were washed three times with buffer I , three times with buffer II  and three times with buffer III . RNA was eluted by phenol/chloroform extraction and ethanol-precipitated and dissolved in 20 μl of water. About 10 μl of immunoprecipitated RNA was reverse transcribed using M-MLV reverse transcriptase (Invitrogen) and random primers (Invitrogen) according to the protocol of the manufacturer. Two microliters of cDNA were subjected to PCR amplification using HPV16-sepcific primer pairs 773S and E1AS, 773S and 438AS or E6F and E6R (Transfected HeLa cells grown in 10 cm dishes were washed by ice-cold PBS followed by crosslinking twice with 0.4 J cm and E6R  as descrFor immunoprecipitation of endogenous ribonucleoprotein (RNP) complexes  from whole cell extracts, HN26 cells or 3310 cells were lysed in cell lysis buffer. The supernatants were incubated with anti-AUF1 antibody  or IgG (Millipore)  overnighON-TARGETplus human hnRNP D siRNA SMARTpool consists of four siRNAs to hnRNPD (L-004079-00-0010) (Dharmacon). A scrambled negative control siRNA (D-001810-10-05) was also purchased from Dharmacon. Transfections were conducted with DharmaFECT1 (Dharmacon) according to the instructions of the manufacturer. The siRNA SMARTpool to hnRNP D or scrambled control siRNAs were transfected in duplicates into C33A2 or SiHa cells grown in 12-well plates for RNA extraction and RT-qPCR or in 6-well plates for protein extraction and western blotting. Cells were harvested at 48 h post-transfection for RNA extraction and RT-qPCR or 72 h post-transfection for protein extraction and western blotting performed as described above.In vitro translation was carried out with TNT(R) Quick Coupled Transcription/Translation Systems (Promega) according to the instructions of the manufacturer. In brief, 100 ng pET32a-HA-E6SDm or 200 ng pcDNA3.1(+)-E1SDm-3XFlag plasmid was translated in the absence or presence of 1 μM recombinant hnRNP D protein (EUPROTEIN) or 1 μM BSA. The 25 μl reactions were incubated for 90 min at 30°C. The translation reactions were analyzed by western blotting as described above. The Luciferase control RNA was also translated in the absence or presence of 1 μM hnRNP D or 1 μM BSA. Luciferase activity in the translation reactions were monitored according to the instructions of the manufacturer using Tristar LB941 Luminometer. Recombinant hnRNP D and BSA were separated on SDS-PAGE followed by staining with Colloidal Blue Staining Kit (Invitrogen).The softeware used to determine band intensity in western blots and RT-PCR gels is ‘Image Lab 6.1.0’ and quantitations were performed with the software ‘Prism GraphPad 8.4.0’.To enhance our understanding of the regulated expression of the HPV16 early genes, we wished to identify hnRNP proteins that control splicing of HPV16 mRNAs. We therefore utilized subgenomic HPV16 reporter plasmid pC97ELsLuc that encodes all HPV16 genes and can produce all alternatively spliced HPV16 mRNAs. The schematic representation of the HPV16 genome Figure , pC97ELsWe transfected plasmids expressing all four isoforms of hnRNP D  Figure  with HPVNext, we investigated if the effect of hnRNP D proteins on HPV16 intron-retained E1 mRNA production could be reproduced independently of upstream splice sites located in E6/E7 gene region. To this end, we used pBELsLuc Figure  that encIn an effort to elucidate how hnRNP D40 inhibits HPV16 mRNA splicing, we investigated the effect of deletion mutations in hnRNP D40 Figure . AnalysiCo-transfection of HPV16 reporter plasmid pC97ELsLuc with hnRNP D40 or either of the N-terminally deleted hnRNP D40 plasmids  Figure  revealed′-splice site SA526 (226∧526)  Figure , while r) Figure ,E. These) Figure ,H and al) Figure ,I but ga) Figure . To dete) Figure : D1 and ) Figure . RRM1 an) Figure , whereas) Figure . We concIn contrast to N-terminal deletions of hnRNP D40, either of the C-terminally deleted hnRNP D40 plasmids D3, D4, D7 and D9 Figure  promotedIn an effort to understand how hnRNP D40 interacted with the splicing machinery, we utilized hnRNP D40 mutants D1 and D2 for further mutagenesis. D1 and D2 have N-terminal deletions but retain either both RRM1 and RRM2 (D1), or only RRM2 (D2). Both D1 and D2 contain an intact C-terminus with the Q-rich region and the RG/RGG-rich ‘RGG’-region Figure . We sepaTo determine if hnRNP D40 interacted with the splicing machinery, we investigated if hnRNP D40 co-immunoprecipitated components of the cellular spliceosome. HeLa cells were transfected with plasmids expressing full-length (D40) or D1 or D2 mutants of hnRNP D40. Cell extracts were prepared and subjected to immunoprecipitation by anti-FLAG antibody followed by western blotting with antibodies to spliceosome components U1-70K, U2AF65 or U2AF35, or with anti-FLAG antibody. Our results revealed that wild-type hnRNP D40 co-immunoprecipitated cellular U1 snRNP component U1-70K and U2 auxiliary factors U2AF65 and U2AF35 Figure . The twocrosslinking and Immunoprecipitation (CLIP) with affinity purification of FLAG tagged hnRNP D40-RNA complexes. FLAG-antibody-immunoprecipitated UV-crosslinked RNA was extracted and subjected to RT-PCR with HPV16 specific primers  interacted with HPV16 RNA. Plasmids expressing FLAG-tagged hnRNP D40, D1 or D2 were co-transfected with HPV16 reporter plasmid pC97ELsLuc into HeLa cells followed by UV-s Figure . The ress Figure . Analysis Figure . Deletios Figure . We conc′-splice site SD226 and 3′-splice site SA409 pulled down hnRNP D40 . To thi′splice site SD226 was mutationally inactivated, plasmid pXH856SDmF   . This trin (BSA) .The results presented above suggested that hnRNP D not only inhibited HPV16 mRNA splicing but also might have the ability to accompany the HPV16 mRNAs to the cytoplasm. As already shown in Figure P = 0.003). Furthermore, KD of hnRNP D resulted in an increase of HPV16 E7 protein levels in SiHa cells . Th. Th43). s Figure , which is Figure . Taken t′- splice site SD3632 that is exclusively utilized during late stages of the HPV16 life cycle, is suppressed in mitotic cells by binding of hnRNP D proteins to AUAGUA motifs upstream of SD3632  (UUAGAUU) partially overlaps with one of the binding sites for hnRNP D identified by PAR-CLIP (UUUAGA). We did not investigate if hnRNP D does bind to the multiple HPV16 RNA sequences directly, but our CLIP experiment showed that hnRNP D binds directly to HPV16 mRNAs in transfected cells. However, hnRNP D probably interacts with HPV16 mRNAs both directly and indirectly via other RNA-binding proteins. It is still unknown if each hnRNP D isoform differ in binding to specific RNA sequences. It would be of interest to use PAR-CLIP to determine potential binding sites for hnRNP D on the HPV16 mRNAs.In addition to the hnRNP D functional analysis, we explored hnRNP D target sites on HPV16 E6/E7 coding RNA sequence using RNA-mediated, protein pull-down assay and found that hnRNP D was associated with the E6 intronic region (nt243-445) as well as with previously identified HPV16 E6/E7 splicing silencer element located between HPV16 nt579-620 . In addis (AREs)  to affecs (AREs)  and reces (AREs) . These is (AREs)  was alsoWe showed that hnRNP D40 inhibited HPV16 early mRNA splicing and increased intron retention on 16E6 and 16E1 mRNAs and accompanied these mRNAs to the cytoplasm. This appears similar to the function of the human immunodeficiency virus (HIV) Rev protein that induces nuclear export of unspliced HIV-1 transcripts to the cytoplasm, thereby upregulating HIV Gag, Pol and Env protein levels . Here we′-non-coding region (NCR) or at the 3′-NCR, resulted in inhibition of translation   and cytonslation  or decrenslation , respectnslation ,69. Intenslation . In concIn contrast to the RNA viruses described above that replicate to high levels and cause acute infections, HPVs establish persistent infections that normally last for 12–24 months, thereby requiring elaborate immune evasion mechanisms, suggesting that the inhibitory effect of hnRNP D on the translation of the E1 and E6 mRNAs may contribute to persistence in the presence of a functional immune system of the host. At the regulatory level, the ratio of E1 to E2, produced from intron-retained and spliced mRNAs respectively, may affect various steps in the HPV life cycle. While higher levels of E1 than E2 is required for genomic DNA replication, increased E2 levels will eventually shut down the early HPV promoter to allow for a re-entry into the cellular differentiation program and completion of the HPV life cycle. Regarding E6 and E7 proteins that are produced from intron-retained and spliced mRNAs respectively, an optimal ratio of E6 and E7 is required to achieve an optimal balance between antiapoptotic and promitotic functions exerted by E6 and E7, respectively. Although it remains to be determined how the intron-retained HPV E1 and E6 mRNAs are translated, hnRNP D may contribute to the modulation of HPV16 mRNA translation and to the control of the delicate balance of the E1/E2- and E6/E7 protein ratios during HPV16 infection.in vivo. Furthermore, we demonstrated an association between endogenous HPV16 RNA and hnRNP D proteins in HN26 cells, a cell line recently isolated from a male patient with HPV16-positive tonsillar cancer, and in the previously described HPV16-immortalized human keratinocyte cell line 3310 , indicating that hnRNP D is available to HPV16 in its natural environment as well as during progression to cancer. It has been shown that the levels of multiple cellular RNA-binding proteins are altered or differ in functional activity through cervical cancer development caused by HPV infection (Finally and importantly, the inhibitory effect of hnRNP D40 on HPV16 E6 or E1 mRNA splicing was reproduced with episomal genomic HPV16 DNA in human primary keratinocytes. This is of significance since the HPV16 genome normally exists as an episome during its life cycle in infected cells 0 Figure . Interesnfection , underscnfection  supportigkac132_Supplemental_FileClick here for additional data file.


Type 2 diabetes (T2D) is a deficiency in how the body regulates glucose. Uncontrolled T2D will result in chronic high blood sugar levels, eventually resulting in T2D complications. These complications, such as kidney, eye, and nerve damage, are even harder to treat. Identifying individuals at high risk of developing T2D and its complications is essential for early prevention and treatment. Numerous studies have been done to identify biomarkers for T2D diagnosis and prognosis. This review focuses on recent T2D biomarker studies based on circulating nucleic acids using different omics technologies: genomics, transcriptomics, and epigenomics. Omics studies have profiled biomarker candidates from blood, urine, and other non-invasive samples. Despite methodological differences, several candidate biomarkers were reported for the risk and diagnosis of T2D, the prognosis of T2D complications, and pharmacodynamics of T2D treatments. Future studies should be done to validate the findings in larger samples and blood-based biomarkers in non-invasive samples to support the realization of precision medicine for T2D. Type 2 diabetes (T2D) remains a significant clinical burden worldwide. Approximately 462 million individuals (6.28% of the world’s population) lived with T2D in 2017, and the prevalence continues to rise . Early dThese goals can be achieved by identifying T2D biomarkers for each purpose. A biomarker is a biological observation that predicts an important endpoint or intermediate outcome in the clinical diagnosis . The BioNumerous studies have been conducted to identify T2D biomarkers. Several reviews have summarized genetic and non-genetic susceptibility/risk biomarkers for developing T2D ,16,17. AThe science of genomes, or “genomics”, studies the structure, expression, and function of the whole DNA sequences of an organism. It has rapidly expanded towards a more functional level, studying the evolution, mapping, and editing of genomes . GenomicAmong all sample types, blood biomarkers represent the most accessible and most studied, given their properties and ease of collection in clinical practice . A wide KCNJ11, PPARG, and TCF7L2 [Heritability is a strong predictor of T2D (26–69% depending on the age of onset), thus motivating the search for genetic predictors of T2D . One wayd TCF7L2 . Two yead TCF7L2 ,28. In 2d TCF7L2 , with and TCF7L2 . In 2016d TCF7L2 . Recent d TCF7L2 .SHBG needs further examination [UGT1A1 locus, are associated with the risk of T2D, making it a candidate as a risk biomarker [Besides using SNPs in a PRS, some studies demonstrate other ways of utilizing SNPs as risk biomarkers. Ding and colleagues demonstrate that a low circulating level of sex hormone-binding globulin is a potential risk biomarker of T2D. Nevertheless, the clinical use of mination . In 2015iomarker . Two yeaiomarker .LRRK2/MUC19 region, indicating that mitochondrial dysfunction is intimately linked to T2D prediction, therefore being a candidate biomarker [One of the first studies researching predictor biomarkers of T2D was done in 2010. In the study, the levels of plasma GAPDH, representing total cell-free DNA, were measured. The levels of cell-free GAPDH were significantly higher in the plasma samples of T2D diabetic patients, becoming a biomarker candidate . The mosiomarker .Urine samples have several advantages above blood: the collection is easy and non-invasive, and the samples are available in large volumes. Anyone can collect urine samples, unlike blood samples which require clinical personnel . Urine iAll T2D genomics urine studies looked into diabetic kidney disease (DKD), including diabetic nephropathy (DN) . DKD is WFS1 gene is associated with a higher estimated glomerular filtration rate (eGFR) in T2D patients with increased albuminuria. This result suggests a potential role of WFS1 as a risk biomarker of T2D and its kidney complications [PGC-1α gene, a genotype previously related to nephropathy in T2D patients. The genotype was found to be associated with a 70% increase in urine albumin excretion in T2D patients with proteinuria compared to T2D patients with normoalbuminuria, making PGC-1α a candidate prognostic biomarker [Two studies found associations between SNPs measured from blood samples with urine proteins. A prospective study reported that rs10010131 in the ications . The seciomarker .The only T2D study on urine-extracted DNA looked into mtDNA from healthy controls and T2D patients with and without proteinuria. Plasma and urine mtDNA content significantly differed between T2D patients and controls, where a reduction in plasma mtDNA content and increased urinary mtDNA/creatinine ratio were observed. The study did not report significant differences between T2D patients with and without proteinuria. So, the findings suggest that mtDNA could be a diagnostic biomarker of T2D .CHGA from peripheral blood leukocytes and measured CHGA concentration in saliva -correlated genes [BSP mRNA, a marker of bone metabolism [Studies on T2D patients with complications have focused on profiling genes related to T2D, inflammation, or specific diabetic complications ,69,70,71ed genes . AnotherSOD1 expression in leukocytes [One study identified a circulating biomarker for treatment response. A clinical trial administered placebo or zinc supplementation in overweight T2D patients and profiled ukocytes . The mRNTTP and inflammatory markers IL-6 and IL-18. TTP encodes tristetraprolin, which is an anti-inflammatory protein. A significantly reduced TTP expression level was found in T2D patients, and the levels are further reduced in patients with macroalbuminuria. In contrast, IL-6 and IL-18 were significantly elevated in T2D patients, especially those with complications [Similar to genetics studies, urinary mRNA studies have compared T2D patients with and without DKD . The firications .NPHS1, NPHS2, and PODXL [Another study looked into podocyte-associated markers: nd PODXL . Renal pnd PODXL .UMOD, SLC12A1, NDUFB2, OAZ1, PPARGC1A, NFE2L2, CD24, SMAD1) using qRT-PCR. So, this is the first agnostic urinary mRNA study where markers were not selected from previously available knowledge. Among the eight mRNAs, UMOD and SLC12A1 levels were elevated in T2D patients with complications compared to those without complications and healthy controls. Significant increases were also seen in NDUFB2 and OAZ1 in T2D patients without complications compared to healthy controls. The four mRNAs were significantly correlated to HbA1c%, but only OAZ1 was correlated to T2D duration. A multi-gene signature was also developed using the eight mRNAs and linear discriminant analysis with cross-validation. The classifier distinguished severe DKD patients from T2D with an AUC of 0.90 [A study with 242 participants used the presence of albuminuria and eGFR to define their study groups . This st of 0.90 .KRAS, SAT1, SLC13A2, and TMEM72 and reduced EGFR and PSMB2 gene expressions [KRAS, SAT1, EGRF, and PSMB2) discriminated T2D patients from healthy controls . So, salivary mRNAs are promising T2D biomarkers, but more studies on these samples are needed.Unlike genetic T2D biomarkers, there is limited research for mRNA biomarkers from saliva samples. One study reported differential salivary gene expression from 13 T2D patients: elevated ressions . The sixEpigenomics studies the whole gene regulation of an organism or cell, including DNA methylation and microRNAs (miRNAs) ,76. EpigThere is growing interest in identifying circulating epigenetic biomarkers for T2D, especially since T2D has a substantial environmental influence, such as sedentary lifestyle and obesity. Identifying risk, predictive, monitoring, and response biomarkers are among the most common research aims in T2D epigenomic studies.All of the T2D DNA methylation blood studies were done on peripheral blood. The bisulfite conversion technologies vary; the most used were: EZ-DNA Methylation Kit from Zymo Research and EpiTect Bisulfite kit from QIAGEN. The DNA methylation technology most used in the studies was Illumina Human-Methylation450 BeadChip, followed by Qiagen PyroMark Pyrosequencing.ABCG1, TXNIP, SREBF1, and PHOSPHO1. With six [ABCG1 is involved in cholesterol and phospholipid transport and insulin secretion. DNA methylation at ABCG1 has been associated with fasting insulin and HOMA-IR [TXNIP is a component of pancreatic β-cell biology, energy metabolism, and cellular redox regulation. TXNIP downregulation protects against obesity-induced diabetes by preventing β-cell apoptosis and preserving β-cell mass [SREBF1 is activated by insulin and contributes to dyslipidemia and hepatic steatosis that occurs in obesity and T2D [PHOSPHO1 is considered to be an attractive target for cardiovascular therapy. It has been found that DNA methylation at this gene is correlated positively with HDL levels [The study designs are different; nevertheless, there are CpG sites in specific genes repeated in several DNA methylation studies . The T2DWith six ,83,84,85With six ,81,84,86With six ,84,85, aWith six ,83 studi HOMA-IR ,88. The ell mass ,90. The  and T2D . The genL levels . The resTFAM [FTO [IGFBP-1 [PRKCZ [TCF7L2 [LINE-1 levels after global DNA methylation were associated with a higher risk of a poor carbohydrate metabolic status. This biomarker could be considered a risk factor of T2D and related metabolic disorders, independent of other established risk factors, therefore having a potential role as a possible biomarker [SLC30A8 [TXNIP [TXNIP [MSI2 gene, and the other one in the CXXC4 gene [PEG3 DNA methylation levels in peripheral blood leukocytes in T2D patients, and four CpG loci were differentially methylated between groups of participants. As a result, these loci might serve as biomarkers of T2D, although additional research is required to strengthen these observations further [ELOVL5 expression is associated with T2D [ELOVL5 gene as a novel epigenetic mark in an epigenome-wide analysis of the blood DNA methylation using T2D-discordant monozygotic twins. The results implied that epigenetic alterations in ELOVL5 demonstrate DNA methylation and RNA expression changes. These provide insights into epigenetic biomarkers for T2D [There are other DNA methylation studies of CpG sites that were reported. There were five publications during the 2010–2013 period, the first one discovered three CpG sites in the gene TFAM , the secFAM [FTO , the thi[IGFBP-1 , the fou1 [PRKCZ , and the [TCF7L2 . In 2014iomarker . From 20[SLC30A8 , the sec8 [TXNIP , the thiP [TXNIP , and theXC4 gene . At the  further . Previouwith T2D ,105. In  for T2D .These methylation markers’ risk or diagnostic value may be small to justify them as a novel biomarker. Thus, further improvement should include CpG sites showing a higher methylation difference.Most T2D miRNA studies were done on blood samples, predominantly plasma, serum, or exosomes . A few sDespite the different study designs, there are commonly reported miRNAs. The most studied and promising T2D biomarker is hsa-miR-126. This miRNA is highly enriched in endothelial cells and is important for angiogenesis and wound repair . It is dOther miRNAs that were reported in at least four studies are hsa-miR-122-5p ,125,126,While most studies used univariate analysis to identify biomarkers, some used multivariate analyses to develop classifiers. For example, a logistic regression model with hsa-miR-148b, hsa-miR-223, hsa-miR-130a, and hsa-miR-19a was able to differentiate T2D patients from those with impaired glucose regulation . The same model had a similar performance in a validation set of 87 patients with impaired glucose tolerance and 113 T2DM patients  . PolygenThe discovery of biomarkers in DNA methylation using urine samples in diabetes is currently an interesting topic of research. Cell type-specific DNA methylation patterns have been widely studied and used to calculate proportions of particular cell types related to diabetes in blood but not in urine . StudiesT2D urine miRNA studies mainly studied T2D patients with kidney complications ,183,184,NFR1 a target for further exploration as a biomarker [DNA methylation studies using saliva samples have been more frequent in recent years. Despite this, DNA obtained from saliva is a combination of bacterial and human DNA. Hence, there are unique challenges in using DNA from saliva samples in methylation studies that can affect data quality in epidemiological studies, such as T2D . Currentiomarker .There is only one salivary miRNA study for T2D biomarkers. The study evaluated four miRNAs that have been reported in T2D and periodontal diseases: hsa-mir-146a, hsa-mir-146b, hsa-mir-155, and hsa-mir-203 . These mBiomarker overlap is mainly seen in DNA methylation and miRNA studies . Twelve SOS2, while other miRNAs target up to 44 genes within the pathway (For circulating miRNA studies, 21 diagnostic miRNAs were reported in at least two studies . Accordi pathway . Althoug pathway .Lastly, more research on non-invasive biomarkers is needed, especially on T2D patients with no complications. Non-invasive biomarkers would be better for screening and diagnosing T2D in many people. Validation studies are also necessary to confirm the utility of these biomarkers. Nevertheless, these studies have identified potential T2D biomarkers that could help guide future T2D studies.


In Mtb group. Please see the correct In S7 FigMtb as shown in Fig 1 and described in the methods section. One, three and five months post Mtb infection lung single cell suspension was prepared and flowcytometry was performed. A representative flow cytometry figure for IL-22 producing (A) LTi and (B) NCR+ ILC3s is shown.Control C57BL/6 and T2DM mice were infected with (TIFF)Click here for additional data file.