E. coli. Bacteriophages were isolated and enriched from sampled farms to study its efficacy to control single and mixed infections with E. coli and IBV in vivo. Twelve out of forty farms were positive for IBV. Phylogenetic analysis of partial spike protein revealed that all positive cases clustered within the GI-23 genotype. Eight out of forty farms were positive for E. coli serogroups O26, O78, O86, O114, O119, with O125 found on three farms. Bacteriophage treatment delayed the onset and reduced the severity of clinical signs, and prevented the mortality associated with single and mixed infection with IBV and E. coli. Furthermore, in mixed infections, bacteriophage treatment significantly reduced E. coli as well as IBV shedding. Groups treated with bacteriophages showed a significant reduction of E. coli shedding that gradually decreased over time, in contrast to higher and gradually increasing shedding without bacteriophage treatment. In conclusion, bacteriophage treatment significantly reduced the pathogenicity and shedding of IBVand E. coli in mixed infections.Infectious bronchitis virus (IBV) represents a major threat to poultry production worldwide particularly when complicated with bacterial infection. In the present study samples were collected from forty broiler farms with respiratory manifestations to characterize IBV and  Mycoplasma gallisepticum (MG), and avian pathogenic E. coli (APEC). Avian respiratory tract infections can be caused by single pathogens or in combination with each other in form of preceding, concurrent or secondary infections [Avian respiratory tract infections are associated with massive economic losses particularly under poor intensive rearing conditions in winter seasons. Several pathogens, adverse environmental conditions and poor managemental factors are involved –3. Pathofections . Change fections , mechanifections .APEC and IBV infections are widely observed under field conditions and are reproduced under experimental conditions resulting in enhanced severe colibacillosis [In Egypt, respiratory diseases outbreaks caused significant economic losses in commercial poultry production during recent years . Laboratcillosis  and enhaUnder field conditions, commercial broiler farms with inadequate biosecurity measures and with the presence of colibacillosis in early age bird are associated with more severe IBV infections in late age birds (>20 days old). Despite the wide spread use of antibiotics and vaccination against IBV, complications as a consequence of mixed infections of avian bacterial pathogens including APEC and IBV became a general phenomenon associated with significant economic losses.In vivo study was conducted to evaluate the efficacy of bacteriophages in reducing the pathogenicity of single and mixed infections with APEC and IBV.In the present study samples were collected from broiler farms with birds with respiratory manifestations. All samples were tested for the presence of IBV and APEC. Recovered IBV isolates were genetically characterized by partial spike protein sequencing. Recovered APEC were characterized by serotyping and antibiotic profiling. Bacteriophages were isolated from water samples collected from the same farms and enriched against APEC O78. g for 5 min. Supernatant was collected and stored at −80ºC until further investigation.One hundred recently succumbed chickens suspected of being IBV infected were collected from forty broiler farms. The study was conducted between late 2017 and early 2018 and samples collected in northern Egypt (Dakahila and Damietta governorates). Lung, trachea, kidney, and liver samples were aseptically collected. On the same day of sampling, half of the organs were cultured for bacteriological investigation in buffered peptone water at 37ºC for 24 h. For IBV screening, lung and trachea samples were homogenized with an equal mass of phosphate buffer saline and then centrifuged at 7,000 × http://www.ncbi.nlm.nih.gov). The sequences were aligned using multiple alignment MAFFT version 7 . The tree was constructed with the MEGA 6 software [http://tree.bio.ed.ac.uk/software/figtree/).Viral RNA was extracted using the QIAamp viral RNA mini kit (QIAGEN) in accordance with the manufacturer instructions. One-step RT-PCR was performed for IBV screening as previously described . Genotypsoftware  using thsoftware . Then, tE.coli isolation and confirmation was performed in accordance with [coli strains were subjected to serotyping using all available O (O1 to O181) antisera in accordance with [E.coli (serotype O78) was tested for purity using API 20E  and counted in colony-forming units (CFU).For the bacteriological investigation, nce with . Confirmnce with , cross-rOver the surveillance period, sewage water samples were collected from poultry farms and used for the isolation of bacteriophages specific to APEC O78. Samples were centrifuged at 10,000 rpm for 10 min and then filtered through a 0.22 μm filter (Millipore). Bacteriophage purification and enrichment was performed in accordance with . BrieflyRLQP in accordance with guidelines of the ministry of agriculture and land reclamation and ministry of environment of Egypt.Animal experimental designs and procedures were reviewed and approved by E.coli O78 isolate recovered in the present study were titrated by colony-forming units (CFU). Birds were challenged with 108 CFU diluted in 100 µl sterile PBS. The IBV strain IBV-EG/1212B-2012 (accession number: JQ839287) belonging to Egy/Var II [6 EID50 in 100 µl per bird. Previously described primers and probes were used to create a standard curve for absolute quantification of IBV shedding [Pure y/Var II  was usedshedding .ad libitum access to feed and water. One day later, chicks were monitored for the presence of IBV or APEC O78 as previously described and then randomly divided into seven groups. Groups 1, 2, and 3 were treated with bacteriophages by intratracheal inoculation with 108 PFU at 1, 5, 8 and 13 days; whilst groups 4, 5, 6 and 7 were not treated with bacteriophages. Groups 1 and 4 were challenged intratracheally at day 2 with 108 CFU APEC O78. Groups 2 and 5 were challenged intratracheally with 106 EID50 of IBV. Groups 3 and 6 were infected with both pathogens in a 3 days interval. Group 7 was kept as a negative control and received PBS. The experimental design is shown in A total of 70 healthy one-day old chicks were purchased from a local poultry hatchery and housed in isolators with All birds were observed twice daily and clinical signs recorded in accordance with . BrieflyStatistical significances of viral and bacterial shedding between different groups were evaluated using Student’s t-test in Microsoft Excel based on the mean of shedding quantities in three tested birds sampled from each group at each time point.E.coli.Forty broiler flocks, aged between 30 and 40 days and suffering from respiratory affections, from the Dakahlia and Damietta governorate were examined. Unwell birds showed respiratory manifestations visible as nasal discharge, sneezing, gasping and swollen head. In all farms examined unwell birds were isolated and subjected to intense antibiotic treatments. However, clear differences in activity and feed intake remained. Screening of collected samples focused on the detection of IBV and E.coli , five farms from the Dakahlia and three farms from the Damietta governate. Serotyping of the isolated strains revealed the presence of the O26, O78, O86, O114, O119 serogroups, with the O125 serogroup detected on three farms. Disc diffusion tests showed the following resistance rates: 100% to penicillin, 75% to gentamycin and streptomycin, 62.5% to ciprofloxacin, 50% to cefotaxime and 25% to doxycycline. 75% of all isolated strains were multidrug-resistant (MDR).Eight farms were positive for Twelve farms were positive for IBV  determined by real-time PCR, with ten farms from the Dakahlia and two farms from the Damietta governate. Postmortem examination of the recently succumbed birds revealed the presence of caseous plug at the tracheal bifurcation, caseous trachitis, and fibrinous pericarditis, perihepatitis and air sacculitis. For virus isolation, specific pathogen free embryonated chicken eggs were inoculated. Total RNA was extracted from the allantoic fluid and the partial S1 gene of each isolate sequenced. Results obtained revealed that the tested samples clustered within the Egyptian variant group of GI-23 genotype subgroups . NucleotClinical signs observed including gasping, coughing or depression started to appear from three days post-challenge with APEC or a mixed APEC and IBV infection. Bacteriophage treatment delayed the onset of the clinical signs to 6 days post-challenge (dpc) and in addition markedly reduced their severity in both groups . RegardiBacteriophage treatment was not associated with mortality in single APEC or mixed APEC and IBV infected groups. In contrast, birds challenged with APEC alone and mixed APEC and IBV infection without bacteriophage treatment showed a 16% and 29% mortality rate at 8 and 7 days post-infection respectively . BacteriBacteriophage treatment significantly reduced APEC shedding after single APEC or mixed APEC and IBV challenge, with a gradual decrease of bacterial loads in lung tissues over time. In contrast, a non-treated and challenged group showed a significantly higher APEC load with a gradual increase over time especially at 9 and 15 dpc . Interesin vivo, as well as their effect in reducing IBV pathogenicity after mixed APEC and IBV infections.Infectious bronchitis in particular complicated by mixed viral and/or bacterial infections is associated with catastrophic economic losses for the global poultry industry, and a significant threat for food security. Under field conditions complications caused by mixed IBV and APEC infections are linked with with accentuating respiratory signs in addition to airsacculitis, pericarditis and possible perihepatitis . Under eOut of forty farms, IBV was detected on 12 farms . The high incidence of IBV in Egyptian poultry flocks suffering from respiratory manifestations (39–75.6%) has been recorded in several previous studies ,10,30,31E.coli in poultry farms’ environment necessitate the use of antibiotics. However, the constant unwise use of antimicrobials in Egypt led to the high prevalence of multidrug-resistant strains that threaten human health and are associated with economic losses in the commercial poultry sector [E.coli. 75% of them showed resistance to three or more antibiotics which were defined as multidrug resistant. This highlights the importance for the development of alternative therapeutic approaches preventing colibacillosis.In chickens, diseases associated with APEC cause significant economic losses due to mortality, decreased feed conversion rates, carcass contaminations and the high cost of its control and prevention. These losses in addition to the high prevalence of y sector ,37. In tBacteriophage therapy has been proven as an effective tool in the treatment of colibacillosis which initiated in the respiratory tract of chicken –40. The In the present study, the efficacy of bacteriophage therapy in single and mixed infections with Egyptian IBV variant and/or APEC O78 has been evaluated. Results obtained revealed a significant higher persistence of APEC and IBV shedding together with higher morbidity and mortality in the mixed infection group comparing to single infected groups. This finding agrees with several previous studies ,42,43. MIn conclusion, the intra-tracheal administration of a high dose of bacteriophages limited APEC replication in the respiratory tract, and subsequently diminished the consequences of single and mixed infections with APEC and IBV. Bacteriophage treatment represents a promising tool for the prophylactic control of colibacillosis, and minimizes the consequences of mixed APEC and IBV infections.


Kremastochrysopsis austriaca (Chrysophyceae), an Austrian snow alga that is not closely related to any of the ice-associated algae examined so far, also produces IBPs, although their activity was weak. Sequencing the algal genome and the transcriptomes of cells grown at 1 and 15°C revealed three isoforms of a type 1 IBP. In agreement with their putative function, the three isoforms were strongly upregulated by one to two orders of magnitude at 1°C compared to 15°C. In a phylogenetic tree, the K. austriaca IBPs were distant from other algal IBPs, with the closest matches being bacterial proteins. These results suggest that the K. austriaca IBPs were derived from a gene that was acquired from a bacterium unrelated to other IBP donor bacteria and confirm by their presence in yet another alga the essential role of algal IBPs.All ice-associated algae examined so far have genes for ice-binding proteins (IBPs), which suggest that these proteins are essential for survival in icy habitats. The most common type of IBP, type 1 IBPs , is also found in ice-associated bacteria and fungi. Previous studies have suggested that algal IBP genes were acquired by horizontal transfer from other microorganisms (probably bacteria). However, it remains unclear whether this is also the case for algae distantly related to the ones examined so far and whether microorganisms other than bacteria could be the donors. Furthermore, there is only limited evidence that these proteins are expressed at low temperature. Here, we show that  Ice-binding proteins (IBPs) are produced and secreted by many ice-associated microorganisms  from both marine and freshwater environments . Some auChloromonas sp., in which TXT motifs appear to be responsible for ice binding , which was first found in a snow mold  and subs binding .Kremastochrysopsis austriaca, was identified in the Austrian Alps (K. austriaca has at least three isoforms of a type 1 (DUF3494) IBP that appear distant from other type 1 algal IBPs and that were most likely acquired from a bacterial gene. The new results, combined with previous results, also point to a possible difference between marine and freshwater IBPs.Recently, a new species of snow alga, ian Alps . It is a−2 s−1. Small numbers of bacteria and another eukaryote, possibly an amoeba, were observed under the microscope. We estimated that they each accounted for less than 0.1% of the total biomass of the cultures.The cells used in the present study were obtained from a melting Alpine snowfield in Tyrol province, Austria in May 2017. At the University of Applied Sciences Upper Austria, a unialgal although non-axenic strain (DR75b) was established at 5°C in DY-V medium and then simultaneously cultured at 1 and 15°C in aerated 250-ml Erlenmeyer flasks. The 15°C cells were cultured in a walk-in environmental chamber, and the 1°C cells were cultured in a Percival LT36VL culture chamber  inside the environmental chamber. The cells were illuminated for 14 h per day with fluorescent lighting at a flux of approximately 40 μmol photons mAfter allowing non-axenic cultures to adapt to 1 and 15°C for 2 weeks, RNA was extracted from intact cells with a NucleoSpin® RNA Plant kit . RNAs (1 and 15°C) having RNA integrity numbers (RIN) of 8.9 and 8.7, respectively, as measured with a model 2,100 Bioanalyzer (Agilent), were sent to Eurofins  for sequencing. There, poly (A) RNA was isolated  and used to prepare strand-specific cDNA libraries for paired-end sequencing. The libraries were sequenced on an Illumina HiSeq platform, yielding 93.2 million 1°C reads and 86.6 million 15°C reads, each with a length of 151 nt. At the University of Nevada Las Vegas (UNLV), the reads were assembled with CLC Genomics Workbench into contig files of 57.4 and 48.5 Mb, respectively. The 1 and 15°C transcriptomes were submitted to NCBI (Bioproject PRJNA557425).de novo assembly following the manufacturer’s instructions and then assembled with SPAdesTo obtain genomic DNA, intact cells were incubated with hexadecyltrimethylammonium bromide (CTAB) extraction buffer and β-mercaptoethanol at 65°C for 1 h. Proteins and DNA were separated by adding an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1). Nucleic acids were recovered by ice-cold isopropanol precipitation, washed twice with ethanol, and dried in a SpeedVac (Eppendorf™ Concentrator plus) prior to rehydration in nuclease-free water. The DNA was sent to Seqomics Biotechnologia  for sequencing. A mate-paired library was generated using an Illumina Nextera Mate-Paired Kit with insert sizes ranging between 7 and 11 kb. The library was sequenced on an Illumina MiSeq instrument using V2 sequencing chemistry resulting in 38.4 million 250-nt reads (19.2 million clusters of 2 × 250 nt). Raw data were pre-processed for IBP DUF3494 domains were identified with NCBI’s conserved domain databaser/R/L, where r is the number of reads matching IBP nucleotide sequences with e values <1e-20, R is the number of millions of reads in the 1 and 15°C transcriptomes , and L is the length of the gene (from start codon to stop codon) in kb. To obtain r, all three IBPs were used as queries, the combined hits were sorted by e value, duplicates were removed, and the hits were then sorted by IBP number. For comparison, the FPKM values of a reference gene, ribosomal protein L23  values using the formula FPKM = 3 rpl23;  were alsK. austriaca cells from Austria was transported to and cultured at Mariposa, AZ, USA, by Dr. Robert Andersen. Cells were grown at room temperature in DY-V medium and shipped to UNLV, where they were gradually adapted to 2°C in M-1 medium  over the DU3494 domain; Swiss Model Template Library ID 6eio.1; The structure of the DUF3494 domain of ss Model 5. Among Kremastochrysopsis austriaca  (K. austriaca sequence was assembled and submitted to GenBank (acc. no. MN441508). The deduced amino acid sequence matches other stramenopile α-tubulins with e values of 00.The alga was identified as phyceae)  partly oGlaciimonas. The large sizes of the contigs (indicating many reads) indicated that these bacteria, despite their low visibility under the microscope, were well represented in the culture.Other sequences in the transcriptomes and genome indicated the presence of contaminating bacteria and at least one eukaryote, possibly an amoeba. The DNA contig file included several large (>1 Mb) contigs whose genes suggested that they were bacterial. The largest contig (3.4 Mb) appeared to belong to a bacterium of the glacier-associated genus Ice-binding activity in the culture medium was difficult to detect without concentrating the medium. At 100× concentration, and after 2 h at −1°C, the freeze-dried, resuspended spent culture medium caused the appearance of nascent pits on the ice basal plane , top andK. austriaca 1 and 15°C transcriptomes for known ice-binding proteins revealed the presence of genes for several very similar isoforms of type 1 (DUF3494) IBPs. No other genes associated with ice binding, such as genes for ice-nucleating proteins, were found. The 1°C transcriptome contained more than 32,000 unique reads matching the isoforms with e values <1e-20, making it unlikely that the reads were from a contaminating organism. In addition, one of the isoforms was on a DNA contig that included an intron-containing gene (acc. no. MK949445) that closely matched  a stramenopile chaperone gene. Three very similar sequences  were assembled using the transcripts, DNA reads, and sequencing of PCR products and submitted to GenBank . The deduced amino acid sequences of the three isoforms have lengths of 246 or 247 residues and have pair-wise identities between 91.9 and 95.2% (similarities between 95.6 and 97.2%). They have the typical sequence structure of type 1 (DUF3494) IBPs, with an N-terminal signal peptide and a 181-a.a. ice-binding domain (TBLASTN searches of the g domain . The DNAChloromonas brevispina (JR unpublished data) and an Antarctic lake Chlamydomonas . Among algae, the closest matches were an IBP from an Antarctic lake alga of the genus Chlamydomonas (acc. no. ARM65346) . Similar matches were obtained when just the DUF3494 domain was used as the query. This distance from other algal IBPs is reflected in a phylogenetic tree of all known type 1 algal IBPs, in which K. austriaca IBPs are well separated from the others  and lower probabilities of being Gram-negative or Gram-positive bacterial .Many if not most bacterial IBPs have a signal peptide, which raises the question of whether the The abundance of IBP transcripts in the 1 and 15°C transcriptomes showed that the IBP1, 2, and 3 isoforms were strongly upregulated at 1°C: 94, 13, and 66 times, respectively . By contKremastochrysopsis austriaca, isolated from melting alpine snow, have been found to produce ice-binding proteins, and in each case, in multiple isoforms. By contrast, no such genes have been found in the mesophilic algae whose genomes have been sequenced. This includes a temperate marine Chrysophycea alga closely related to K. austriaca (Ochromonas sp. CCMP1393) whose genome contains no IBP homologs. On the other hand, only a small percentage of ice-associated bacteria have such genes  IBPs have multiple effects on ice. Those with a signal peptide, which usually acts as a secretion signal, appear to modify the external environment in two ways, despite their very low concentrations. First, they inhibit the recrystallization (RI) of ice, whose moving grain boundaries are thought to damage cell walls . RI has  glaciei  and Fragylindrus . Second,ylindrus  and a fuylindrus . It has ylindrus . Some IBolerance .K. austriaca are the source of the ice-binding activity as shown in K. austriaca genome or transcriptomes.Although we were unable to directly show that the type 1 IBPs of It is unclear why the IBP activity was weak, despite the high transcript abundance, as media from other cultures of IBP-producing algae  showed aFragilariopsis cylindrus, which has a large family of type 1 IBPs. When F. cylindrus cells growing in marine medium (34 ppt) at 5°C were rapidly transferred to conditions found in sea ice brine pockets , transcript abundance of some IBP isoforms increased between 70 and 200 times after 20 days  (Chloromonas) was found to increase with increasing ice slush content in the medium (The strong upregulation of IBP transcripts at 1°C is consistent with their having a role in mitigating freezing damage. The upregulation is comparable to that observed in the sea ice diatom creased) . IBP procreased) . In addie medium . Eukaryoe medium . On the e medium . This maK. austriaca IBP1 was an IBP isolated from an Antarctic marine bacterium . However, in some cases, the C side and several loop regions (including the α-helix on the A side) have been suggested to also be involved in ice binding . Of nine efcIBP; . The pre efcIBP; . On the  efcIBP; . Among t, do not . K. austng sites . Similariaca IBS .K. austriaca’s IBPs for the same reasons: they are unrelated to the IBPs of other stramenopiles  IBP genes were acquired by horizontal gene transfer (HGT), most likely from bacteria . This alenopiles , and theAntarctomyces psychrotrophicus was found to have two dissimilar DUF3494 type IBP genes that could best be explained by independent acquisition from bacteria .JR and DR contributed equally to this work.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.


Saccharomyces cerevisiae for rapid, specific inactivation of individual kinases by addition of a covalent inhibitor.Cyclin-dependent kinases play multiple roles in RNA polymerase II transcription. Cdk7/Kin28, Cdk9/Bur1, and Cdk12/Ctk1 phosphorylate the polymerase and other factors to drive the dynamic exchange of initiation and elongation complex components over the transcription cycle. We engineered strains of the yeast  Saccharomyces cerevisiae for rapid, specific inactivation of individual kinases by addition of a covalent inhibitor. While effective, the sensitized kinases can display some idiosyncrasies, and inhibition can be surprisingly transient. As expected, inhibition of Cdk7/Kin28 blocked phosphorylation of the Rpb1 C-terminal domain heptad repeats at serines 5 and 7, the known target sites. However, serine 2 phosphorylation was also abrogated, supporting an obligatory sequential phosphorylation mechanism. Consistent with our previous results using gene deletions, Cdk12/Ctk1 is the predominant kinase responsible for serine 2 phosphorylation. Phosphorylation of the Rpb1 linker enhances binding of the Spt6 tandem SH2 domain, and here we show that Bur1/Cdk9 is the kinase responsible for these modifications in vivo.Cyclin-dependent kinases play multiple roles in RNA polymerase II transcription. Cdk7/Kin28, Cdk9/Bur1, and Cdk12/Ctk1 phosphorylate the polymerase and other factors to drive the dynamic exchange of initiation and elongation complex components over the transcription cycle. We engineered strains of the yeast  Accordingly, there is great interest in chemical kinase inhibitors, both for scientific studies and as potential drugs. The greatest challenge is to create compounds with sufficient specificity to overcome the structural similarities among kinase active sites. One powerful strategy is the “bump-hole” system, in which a “gatekeeper” residue in the target kinase ATP-binding pocket is mutated to create space for an ATP mimic carrying a bulky side group that blocks its binding to other kinases  involved in transcription by RNA polymerase II , which has both a “bump” that can be accommodated by a “hole” mutation in the ATP-binding pocket and the CMK functional group that covalently cross-links to an engineered cysteine near the active site , we found that Ser2P was also strongly blocked upon Kin28 inhibition, indicating clear sequential dependence of the two marks. Bur1 inhibition also reduced CTD Ser2 phosphorylation, but less than Ctk1 inhibition, supporting our earlier findings that Bur1/Cdk9 is not the major Ser2P kinase .Inhibition of each kinase gave a different response. Notably, time courses revealed that CMK inhibition of all three kinases was very rapid but transient. For example, a 15-min CMK treatment of Ctk1-IS cells caused very strong loss of Ser2P but no effect on Ser5P, as expected for the major Ser2 kinase. However, Ser2P showed partial recovery by 90 min . As assaΔ strain . InhibitΔ strain  and 4. TAs expected, treatment of the Kin28-IS strain with CMK led to a significant drop in CTD Ser5P. This was seen with both 3E8 and H14 antibodies  and 4. SLike that in Ctk1-IS and Bur1-IS, inhibition of CTD phosphorylations in the Kin28-IS strain was also transient, with recovery apparent by 60 min after CMK addition . This waCYC1 core promoter and a G-less cassette –Rpb1 linker region fusion protein was phosphorylated with either Bur1 or Ctk1 and tested for in vitro binding to the Spt6 tSH2 domains using far-Western blotting . The results showed that Bur1/Cdk9 was the only required kinase. Both T1471 and S1493 phosphorylations were unaffected by Kin28-IS or Ctk1-IS inhibition but were strongly reduced by CMK treatment of the Bur1-IS strain  alleles can be very informative, but products of these genes are often partially defective at permissive temperatures or remain partially functional at nonpermissive temperatures. Degron fusions can support normal function under permissive conditions, but loss of function may be slow or incomplete. Interpreting outcomes from these methods can also be complicated, as loss of the kinase protein can have secondary effects distinct from the loss of the kinase activity, such as destabilization of interacting factors. Chemical inhibitors can overcome these issues, as the protein remains present while kinase activity is ablated. However, this approach requires the availability of an inhibitor that completely inactivates the target kinase without affecting other kinases.To understand the role of an individual kinase, one must observe the effects of inactivating its function in vivo, but effects on protein expression and activity prior to addition of the inhibitor must be monitored.Here we applied the IS allele approach –4 to the–Although covalent modification of the kinases is presumably nonreversible, we found that the effects of CMK inhibition can be transient in yeast liquid cultures. Although growth of Kin28-IS or Bur1-IS on plates was completely arrested on solid medium containing CMK , CMK didThe previous characterization of Kin28-IS by Rodriguez-Molina et al.  nicely din vivo inhibition of Kin28-IS produces strong inhibition of Ser2P, as well as Ser5P and Ser7P. The Ser2P drop was unexpected, as this phosphorylation was reported to be unaffected in multiple earlier studies using AS alleles of Kin28/Cdk7 in budding or fission yeasts  of Spt5, where phosphorylation promotes binding of the PAF complex elongation factor , 37. IntBUR2 -tagged kinases were previously described , 40. Muto method . CorrectPolyclonal antiserum that specifically recognizes Rpb1 pS1493 was previously described . Polyclo595] of ∼1.0) in standard YPD medium , and 80-μl aliquots were prepared. Kinase complexes were prepared as described below, and 1.5 μl of each kinase was added to the MPB-Rpb1 and incubated at 30°C for 1 h. For blotting, 15 μl of reaction mixture was transferred to a tube containing 5 μl 4× SDS-loading dye. Five microliters of each sample was loaded on a 12% gel and electrophoresed for 1 h at 160 V. Far-Western blotting with glutathione S-transferase (GST)–tSH2, followed by anti-GST antibody, were performed as described previously (His-MBP-Rpb1 (residues 1456 to 1511) protein was diluted to 0.5 mg/ml in kinase buffer  were grown in YPD inoculated with 50 ml of a saturated overnight culture and incubated at 30°C until they reached an OD of about 3. Cells were harvested by centrifugation, washed once with cold water, and pelleted by centrifugation. Cells were frozen by passing through a syringe into liquid nitrogen and lysed under liquid nitrogen using a Spex SamplePrep 6870 Freezer/Mill . Pulverized yeast was thawed in 2 pellet equivalents of lysis buffer . Lysates were clarified by centrifugation at 37,000 relative centrifugal force (RCF) for 30 min. The supernatant was incubated with 0.5 ml of Ni-nitrilotriacetic acid (NTA)–agarose (Qiagen) for 30 min at 4°C with agitation, followed by 3 washes with 5 ml of lysis buffer and then 2 washes with 5 ml of wash buffer , and finally eluted with 4 times 2 ml of elution buffer . The eluted protein was incubated with 20 μg PreScission protease in 6,000- to 8,000-molecular-weight-cutoff Fisherbrand regenerated cellulose dialysis tubing in dialysis buffer  overnight. The protease was removed with glutathione-agarose. The subsequent flowthrough fraction was collected, and the resin was washed 2 times with 1 ml of dialysis buffer. The flowthrough and wash were pooled, concentrated to 200 μl using a 30-kDa Vivaspin concentrator , and flash frozen in liquid nitrogen prior to storage at 80°C. The negative-control strain was processed in parallel. Coomassie blue-stained gels of each preparation are shown in Fig. S3A.Wild-type Ctk1 and Bur1 complexes, carrying a PreScission protease-cleavable HisEscherichia coli cells (Stratagene). Cultures were grown in 2 liters of autoinduction medium (GST-Spt6 tSH2 (residues 1247 to 1451) was expressed and purified as described previously  Fig. 5AFig. 5A. n medium  in baffl


To evaluate the therapeutic effect of Shengjing capsules on nonobstructive azoospermia (NOA) in the rat model. n=5 per group): normal group, NOA group, and three Shengjing capsule treatment groups . HE staining and semen smear were performed to assess sperm quality. The expression levels of PI3K/AKT and integrin α6/β1 were measured by qRT-PCR and western blot analyses.  Twenty-five male Sprague–Dawley rats were randomly divided into five groups as follows (P=0.0001). The expression of PI3K/AKT and integrin α6/β1 was scarcely expressed in the NOA group. All indexes mentioned above were significantly different from those of the medium- and high-dose groups . The sperm count of rats treated with Shengjing capsules was significantly higher than that of the NOA group (P=0.0001). The rats of Shengjing capsule groups had more layers of spermatogonial stem cells and spermatocytes, and some had intracavitary sperms.  In the NOA group, almost all of the seminiferous tubules were vacuolated with a thin layer of basal compartment containing some spermatogonial stem cells. The counts of sperms in the NOA group were strongly lower than those of the normal group (α6/β1 expression via the PI3K/AKT pathway. Shengjing capsules may be a promising therapeutic medicine for NOA. The underlying mechanisms might involve activating SSCs by upregulating the integrin  Infertility hampers about 15% of couples attempting pregnancy, and male factor infertility accounts for approximately half of all the reasons of this impaired fecundity . Almost In recent years, many techniques, including spermatogonial stem cell (SSC) transplantation, testis tissue transplantation, and induced pluripotent stem cell and gene therapy, have emerged and developed. Intracytoplasmic sperm injection (ICSI) has also been improved, increasing the chances for men to father a child. However, the effective treatment for NOA patients is absent. Thus, it is urgently needed to find an efficient and feasible therapy.α6-integrin, and β1-integrin are useful immunohistochemical staining markers of cell populations, which are highly expressed in stem cells. Especially, α6-integrin and β1-integrin were recognized as marker molecules on SSCs [NOA is characterized by severely impaired or nonexistent spermatogenesis . This su on SSCs , 9.Cordyceps sinensis, Epimedium, and medlar [Panax ginseng extracts have shown significant improvements in sperm concentrations and motility in semen analysis from a randomized controlled trial [Panax ginseng plays an important role in improving sperm hyperactivation via the cation channel of the sperm protein gene expression. Lycium barbarum (L. barbarum), also named wolfberry, obviously has the protective effect on the spermatogenesis of rats with the impaired reproduction system induced by cyclophosphamide [Epimedium, has been proved to improve endothelial cell function in the penis and promote the formation of NO [Cordyceps militaris extracts significantly enhanced the sperm production at the end of the first month and peaked it at the second month [The use of traditional Chinese herbal medicine to improve testicular spermatogenesis has been in China for more than 2,000 years. Shengjing capsules are made up of a variety of medicinal materials that improve sperm production, such as ginseng, d medlar –14. Panaed trial . Park eted trial  reportedsphamide . Icariinon of NO . Additiond month . Shengjind month –18. The α6/β1 expression by the PI3K pathway to activate SSCs and improve the therapeutic effect of spermatogenic function on NOA rats.Shengjing capsules have the potential to improve NOA spermatogenesis, but their mechanism is not yet clear. We have for the first time explained that Shengjing capsules can upregulate the ∗ 6.2≈504 mg/kg·day). The main active principles of Shengjing capsules are displayed in Shengjing capsule extracts, provided by Liao Yuan He Tang Pharmaceutical Co., Ltd. , were officially approved in the treatment of male infertility and asthenospermia by the State Food and Drug Administration of P.R. China (SFDA) (standard number WS-11457(ZD-1457)-2002-2011Z; national drug approval Z20027672). The Shengjing capsule manufacturer recommends 504 mg/day for pharmacological research in rats. Meanwhile, in the dose conversion between animals and humans, the US Food and Drug Administration guidance for industries  recommenTwenty-five male SPF Sprague–Dawley rats  were purchased from Guangdong Medical Laboratory Animal Center  2013-0093). The animal room was kept under 12-hour light/dark cycles and maintained at 20 ± 2°C with 45%–65% relative humidity. The animals had free access to food and water. All the rats were randomly divided into five groups (five rats each): (1) the normal group: the rats were treated daily with distilled water (2 ml) by oral gavage; (2) the NOA group: the rats underwent an NOA modeling, which was conducted by intraperitoneally injecting busulfan 10 mg/kg on day 1 and day 21, as reported previously [All experimental protocols were subject to approval by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Guangzhou Medical University .To determine the sperm morphology, the animals were sacrificed after anesthesia. Then, the cauda epididymides of all animals were quickly transferred, and epididymal fluid was observed in the polarizing microscope. Then, they were minced in 10 mL prewarmed 0.9% normal saline to incubate at 37°C for 10 minutes that allowed sperm to swim out of the lumen of the cauda epididymides for sperm characteristic analysis.μl drop of caudal epididymal sperm solution was loaded under the cover slip. The haemocytometer was placed under the light microscope and viewed under ×400 magnification. The sperm count was calculated by counting 4 × 4 squares  [The sperm count was determined using the haemocytometer under light microscope. A cover slip was placed on the haemocytometer before a 10 tically) .μm thicknesses. Then, tissue sections were deparaffinized in xylene and hydrated in descending concentrations of ethanol before hematoxylin and eosin (HE) staining. Morphological changes were observed under a microscope, for example, the morphology of convoluted tubules, the presence of vacuolization, and the death and degeneration of SSCs and spermatocytes. For histomorphometric analyses, cells were recorded in 50 random seminiferous tubules to compare the differences in average cell numbers among the five groups.An abdominal incision was made, both sides of testes were dissected out, and part of them were fixed in Bouin's fixative for histological investigations and subsequently embedded in paraffin. The embedded tissues were cut into 4 α6-integrin and β1-integrin mRNA in five groups were assayed by qRT-PCR analysis in accordance with the protocol [Expression levels of protocol . The comα6 , Anti-Integrin β1 , phospho-Akt polyclonal rabbit antibody at a dilution of 1 : 2000 , and phosphoinositide 3-kinase (PI3K) at a dilution of 1 : 2000  overnight at 4°C and incubated with anti-rabbit IgG horseradish peroxidase conjugated secondary antibodies . The relative protein expression was normalized with GAPDH .Total protein was extracted from testicular tissue. Western blot analysis was conducted as previously published by our laboratory . The memα6 , and Anti-Integrin β1 . After stained according to the standard immunohistochemical protocol, immunoreactivity was evaluated by assessing positive cell percentages and staining intensities. The percentage scoring of immunoreactive cells was as follows: 0 (0–5%), 1 (6–25%), 2 (26–50%), 3 (51–75%), and 4 (>75%). The staining intensity was visually scored and stratified as follows: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). A final immunoreactivity score (IRS) was obtained for each case, multiplying the percentage and the intensity score.Testicular tissue section slides in five groups were heated at 60°C for approximately 1 hour in a hot air oven. Then, they were deparaffinized in xylene and rehydrated using alcohol gradient. The antigen retrieval process was performed. Then, they were cooled to room temperature. The primary antibodies used were p-Akt  at a 1 : 200 dilution, Anti-Integrin t-tests. The one-way ANOVA was done for comparison among different groups. P < 0.05 was examined statistically significant.SSPS 13.0 software  was utilized to analyze the experimental data. All experiments were performed more than three times, the data of this research were described as means standard deviations (SDs), and comparisons were performed using Student's P > 0.05). After Shengjing capsule treatment, we weighted body, testicular, and epididymal weights to establish the declining trend of spermatogenesis. As shown in P=0.0001 for both) in comparison with the normal group. In Shengjing treatment groups, the testicular and epididymal weights in the medium-dose group increased significantly  compared to the NOA group. Similarly, there was a significant improvement in testicular and epididymal weights in the high-dose group compared to the NOA group . No significant differences were detected in these data between the NOA group and low-dose group (P=0.957 and P=0.293). No obvious difference in body weight was proved among the five groups. The findings presented here suggested a beneficial effect of Shengjing capsules on spermatogenesis in the NOA rats.The initial body weight did not differ significantly among the five groups . Furthermore, densitometric analysis of the western blotting bands revealed that values of α6-integrin were 0.40, 0.59, 1.23, and 1.27 for NOA and low-, medium-, and high-dose groups, respectively .In this study, quantitative RT-PCR and western blot results showed that al group . In Shenectively . The expectively . Compareα6/β1 expression in the SSCs . Similar results were presented for β1-integrin .To detect the expressions of PI3K and Akt in testes, we first confirmed the expression of phosphorylated protein kinase B (p-Akt) by immunohistochemistry. After Shengjing capsule treatment, an evident expression of p-Akt can be observed in the cells near the basal lamina of seminiferous tubules in rat testes, where SSCs are located . The briPatients with azoospermia can achieve pregnancy by extracting sperm from the testicles through assisted reproductive technology . For NOAIn this study, we used HE staining and semen smear to find that the number of spermatogenic epithelial cells was higher in the Shengjing treatment group, and the sperm count was higher than that in the NOA rat group. It showed the disturbance in the seminiferous tubular layers and the disappearance of spermatocytes and spermatids in the NOA group. Almost all the seminiferous tubules across the section were vacuolated with a thin layer of the basal compartment containing Sertoli cells and some SSCs in HE staining. After 8 weeks of Shengjing capsule treatment, the count of the spermatogenic cells, the germ cells, and even the sperms in the medium-dose and high-dose groups was significantly higher than that in the NOA group. These results indicated that the Shengjing capsule could repair the damaged seminiferous epithelium and protect spermatogenesis.The spermatogenic function of azoospermia rats was improved in the medium- and high-dose spermatogenic capsule groups. However, the spermatogenic function of rats did not increase with the increasing dose of the Shengjing capsule. This is consistent with the clinical use of Shengjing capsules to improve the semen quality of azoospermia patients. These results suggest that Shengjing capsules may have similar effects on improving semen quality in NOA but require more multicenter studies with larger samples.Spermatogenesis is a complex process that originates with and depends on SSCs. Spermatogonial self-renewal is a critical prerequisite for the maintenance of normal sperm counts. And the disruption in their function has dire consequences on fertility. These primitive cells reside on the basement membrane of the seminiferous tubule and slowly proliferate to provide (a) additional stem cells by self-renewal and (b) progeny cells that undergo significant amplification during the differentiation process to spermatozoa . A singlα6/β1 is an important biomarker of SSCs. These effects make it possible for SSCs to support homeostasis and provide sperm regeneration [Stem cells have been reported as a star therapeutic target in a number of cases of busulfan causing NOA model literatures . SSC hasneration .α6-integrin and β1-integrin for SSCs in testicular tissue. We find that the integrin α6/β1 expression is barely expressed in the NOA group. And in medium-dose and high-dose groups, the expression levels of integrin α6/β1 significantly increased compared with the NOA rat group. It means that a significant increase is shown in the numbers of SSCs after Shengjing capsule treatment, though the improvement does not increase with the dose. Since the vertebrate-conserved RNA-binding protein DND1 is necessary for the survival of SSCs, we further examine the DND1 expression. Taken together, these findings reveal that Shengjing capsules might activate SSCs via upregulating integrin α6/β1.In this study, we observe that Shengjing capsules significantly improve the spermatogenesis in the NOA rats. To elucidate the underlying mechanisms, we detect the expression of the specific biomarker of α6/β1. Western blotting analysis demonstrated that PI3K and p-Akt expressions apparently enhanced in medium- and high-dose Shengjing capsule groups compared with the normal group. Immunohistochemistry analysis corroborated these results, showing that expressions of PI3K, p-Akt, and integrin α6/β1 were significantly higher in medium-dose and high-dose Shengjing capsule groups than in the NOA group. PI3K/AKT may play an important role in the improvement of azoospermia when using Shengjing capsules for upregulating integrin α6/β1.The PI3K/AKT pathway might play an impotent role in male fertility . Male miPanax ginseng extract could enhance testicular function by elevating GPx and GST activity, thus resulting in increased glutathione, which prevented LPO in the testis [Panax ginseng extract attenuates H2O2-induced spermatocyte oxidative stress in mouse spermatocytes and regulates the expression of antioxidant-related genes, spermatogenesis-related proteins, and sex hormone receptors in aged rats. Ginseng may be a potential natural drug used to protect from or treat stress-induced male infertility. Zhang et al. [Lycium barbarum could improve sperm density, sperm movement, and the rate of normal sperm morphology. Lycium barbarum could also reduce cadmium-induced testicular damage by increasing antioxidant enzyme activity and reducing oxidative stress. It may be a potential adjunct to testicular toxicity. Taken together with the findings presented here, Shengjing capsules, as a Chinese herbal medicine, may activate SSCs into the process of spermatogenesis by upregulating the expression of integrin α6/β1 through the PI3K/AKT signal pathway in the NOA rats.The Shengjing capsule comprises 19 ingredients, some of which are used to improve testicular function. For instance, the e testis . Kopallie testis  also repg et al.  reportedWhen evaluating spermatogenic function of rats, only the changes of sperm number were evaluated. No other parameters of sperm quality were compared, such as sperm motility and deformity rate. This is due to the lack of professional evaluation of the rat sperm instrument and cannot carry out effective assessment of rat semen quality. Meanwhile, sperm count is one of the indicators of semen quality, and the increase in the sperm count partly reflects the restoration of spermatogenesis in mice. We did not interbreed rats among the treatment groups to evaluate the fertility achieved by the Shengjing capsules in rats. The restoration of spermatogenesis could theoretically lead to the restoration of fertility in mice. More relevant research is needed to clarify the mechanism of Shengjing capsules in improving spermatogenic function in patients with NOA.Traditional Chinese medicine for azoospermia enriches the present-day therapies in the field of sexual medicine. To our knowledge, this is the first study to evaluate the therapeutic effect of Shengjing capsules on NOA. We elaborate a possible potential mechanism and provide a new direction for the treatment of NOA, and further research is needed to clarify the possible mechanism of action. It provides evidence for the clinical treatment of NOA patients using Shengjing capsules.α6/β1 expression via the PI3K/AKT pathway. Our results suggest Shengjing capsules as a promising therapeutic medicine for NOA.This study firstly shows that the Shengjing capsule significantly improves the spermatogenesis in the NOA rats. The underlying mechanisms might involve activating SSCs by upregulating the integrin


Methods of testing and describing the recrystallization process in ice cream systems were characterized. The scope of this study included a description of the recrystallization process and a description and comparison of the following methods: microscopy and image analysis, focused beam reflectance measurement (FBRM), oscillation thermo-rheometry (OTR), nuclear magnetic resonance (NMR), splat-cooling assay, and X-ray microtomography (micro-CT). All the methods presented were suitable for characterization of the recrystallization process, although they provide different types of information, and they should be individually matched to the characteristics of the tested product. The most important factor that determines frozen food quality is the course of crystallization . CrystalIce cream is a multiphase physicochemical system originating from the dispersion of individual components in different phases ,12. The The recrystallization process occurs at a constant temperature during long storage, especially above the glass transition temperature ,17,18. HHydrocolloid stabilizers are used in food production to modify water-binding capacity, freezing rates, ice crystal formation, and rheological properties ,11,12,24Different forms of carrageenan are commonly used as stabilizers. The kappa carrageenan form is mostly used to stabilize dairy products, but it may also be applied to control crystal growth in sorbet production ,25. The The aim of this review was to outline the basic characteristics of the measurement method, sample preparation, and equipment required to show and describe ice crystals during and after the recrystallization process using the following methods: FBRM, OTR, NMR, splat cooling, microscopy analysis, and X-ray microtomography.Control of the ice crystallization process is mostly conducted in an empirical way, mainly due to a lack of experimental data. Research on ice crystal size distribution (CSD) is not simple, especially because of the possibility of melting under unstable conditions and also when it is about a type of ice cream product that contains three phases . The focThe real-time particle size analysis technique of FBRM works by focusing a laser beam directly down the probe tip through a sapphire window. The optical part is rotated about an axis to the probe (2 m/s), so that the beam traces out the circular path (reflected light is detected in the probe). The probe tip is inserted, at an angle, directly into the process streams, to ensure that particles can flow easily across the probe window where the measurement takes place . The lasGenerally, the FBRM instrument can acquire thousands of chord lengths per second. On a counter board, these lengths are classified into a series of size ranges that are expressed as a distribution, referred to as a chord length distribution (CLD). This technique considers the shape and dimensions of the particles. However, the CLD value does not gives information about morphology of the particles and it is less useful for the characterization of a crystal lattice in frozen products ,7,27.Amamou et al. (2010)  presenteArellano et al. (2012)  demonstrThe recrystallization process in ice cream using the FBRM technique was investigated by Ndoye and Alvarez (2015) . They coThe main advantage of the FBRM technique seems to be its suitability for on-line measurements of high solid-concentration suspensions and for following rapid crystallization kinetics. However, it only provides information about the total number of ice crystals and the changes of diameters of ice crystals, and therefore the shape and the changes of ice crystal location are measured.It was shown that the dynamics of rheological measurements can be used for characterization of textural properties and structure of foamed dairy emulsion. The dynamic storage modulus G’ (elastic response) and loss modulus G” (viscous behavior) can provide information about properties of viscoelastic materials.Stanley et al. (1996)  proved tWildmoser et al. (2004)  used rheEisner et al. (2005)  examinedSensory quality closely correlates with viscoelastic properties of products. Tsevdou et al. (2015)  correlatContent, purity, and molecular structure of a sample can be determined using an analytical chemistry technique called nuclear magnetic resonance (NMR). When the NMR technique is used it is possible to quantitatively analyze mixtures containing known compounds. For unknown compounds, NMR can either be used to match against spectral libraries or to infer the basic structure directly. The NMR technique can be used to determine molecular conformation in solution and to study physical properties at the molecular level such as conformational exchange, phase changes, solubility, and diffusion. In order to achieve the desired results a variety of NMR techniques are available. In addition, NMR is versatile and has the potential to be nondestructive, which makes it a potential tool in quality control of various products, including milk-based desserts .The principle of the method relates to the fact that many nuclei possess spin and all nuclei are electrically charged. If an external magnetic field is applied, energy transfer is possible from the base energy to a higher energy level . The energy transfer takes place at a wavelength that corresponds to the radiofrequencies, and when the spin returns to its base level, energy is emitted at the same frequency. The signal that matches this transfer is measured in many ways and processed in order to obtain an NMR spectrum for the charged nucleus ,37.The NMR technique has been shown to be an appropriate method to calculate the amount of unfrozen water in a food sample. Lucas et al. (2004)  examinedHagiwara et al. (2006)  investigIt has been discovered that NMR could be used to determine fat globule size in ice cream and to determine the effect of the formulation on hard ice creams’ structure ,41,42. LThe NMR technique does not involve any thermal processes to assess the amount of ice and thus can be performed at a stable temperature. It is also important that the relaxation parameters of water were used to provide information on the water/non-water molecule interactions. This technique may be a valuable tool for understanding how various stabilizers affect the three-dimensional vein network and recrystallization processes.In order to determine the ice recrystallization inhibition (IRI) activity of ice cream stabilizers, the capillary method or splat-cooling assay can be performed . AlthougThe IRI activity of different substances such as AFP and some polysaccharides can be demonstrated using “splat” assay. This is not a new method and has been used for nearly twenty years. In these experiments, a small volume of sample was usually expelled from a height of 1.5 to 3 m onto a metal plate  that had been cooled in liquid nitrogen or dry ice. The sample drop froze upon hitting the metal plate, forming a thin (splat) wafer of ice. This wafer was later transferred to a microscope stage at a high sub-zero temperature where the sample recrystallized over time and annealing temperature . Different modifications of this method are possible, for example, the sample could be snap frozen between two cover slips. This technique was complemented by crossed polarizers with a dissecting microscope and ice-binding activities of some substances that were examined by Raymond and Knight (2003) . The splThe most popular and universal method of testing and describing the recrystallization process is direct light microscopy observation followed by image analysis of the ice crystals. When the work was started with another technique such as oscillatory thermos-rheometry or splat cooling assay, to complete it the work was usually supported by an appropriate microscopy technique, a cryo-scanning electron microscope or a microscope with a polarizer ,44. PhysDonhowe and Hartel  ,17 exami50) and the slope of cumulative distribution at X50. The recrystallization rate was calculated as the slope of linear regression of the curve plotted with values of X50 for each cycle. Using this method, they proved there was significant retardation of recrystallization with the addition of sodium alginate and xanthan. The same technique  was first used to explain the IRI activity of kappa carrageenan hydrolysates in model sucrose solutions [Regand and Goff (2003)  presenteolutions  Table 150 and thThe method described above makes it possible to analyze not only the changes in diameter of ice crystals but also their shape and location. On the basis of the images it is possible to analyze the mechanism of recrystallization. From the shape of the ice crystal, it is sometimes easy to read that accretion between adjacent crystals occurred . NumerouWhen the growth rate of ice crystals in samples with the IRI active substances is small, there is no difference in morphology or size of ice crystals, as was observed by Kamińska-Dwórznicka et al. (2016)  for kappThe technique of microscopy and image analysis has brought positive results in ice crystal microstructure investigations. However, it is a two-dimensional technique and it does not characterize multidimensional structure of ice cream mix before and after freezing. During the recrystallization process, the microstructure of ice cream  changed The micro-CT method is based on the same assumptions as classic tomography. However, by using a smaller radiation spot, it is possible to obtain a higher resolution of the reconstructed image ,58. ThisGuo et al. (2017)  presenteThe technique of microscopy and image analysis allows one to describe ice crystal microstructure. From the images of the ice crystals we can easily obtain information about the size and the changes of their shapes and location during storage at different temperature and time conditions. The images can be easily analyzed using specific computer software. The main disadvantages of this method are difficulties in the preparation of samples and its influence on the repeatability of results. The technique of X-ray microtomography seems to offer a new possibility in the analysis of the recrystallization process as a non-destructive method that shows ice cream samples in 3D, but has some difficulties with the final interpretation of images. The FBRM (focused beam reflectance) technique is fully automated and provides results more easily and faster than simple image microscopy and image analysis. It is a suitable method for in situ measurements, and it allows sample preparation to be avoided because the measurement is conducted by the probe immersed in the ice cream mixture. However, it can only provide information about changes in the diameters of crystals, without shape and location analysis. The OTR (oscillatory thermo-rheometry) technique is a method in which viscoelastic properties of ice cream closely correlate with the sensory quality, and hence it provides information about the shelf-life of ice cream without recrystallization changes. However, the changes in size or shapes and location of ice crystals are not measured. Nuclear magnetic resonance (NMR) is an effective method to evaluate the amount of unfrozen water in a food sample. Hence, it is a valuable tool for understanding the impact of a stabilizer on the recrystallization processes without providing any information about sizes of ice crystals and locations. Splat-cooling assay is the oldest method to describe the amount, sizes, and morphology of ice crystals. It is a method with a very specific technology for sample preparation and is not suitable for different types of frozen food.All of the discussed methods are suitable for describing the recrystallization processes, although they provide different types of information, and they should be matched individually to the characteristics of the tested product.


In mammals, motility and fertilizing ability of spermatozoa develop during their passage through the epididymis. After ejaculation, sperm undergo capacitation and hyperactivation in the female reproductive tract – a motility transition that is required for sperm penetration of the egg. Both epididymal initiation of sperm motility and hyperactivation are essential for male fertility. Motility initiation in the epididymis and sperm hyperactivation involve changes in metabolism, cAMP (cyclic adenosine mono-phosphate), calcium and pH acting through protein kinases and phosphatases. Despite this knowledge, we still do not understand, in biochemical terms, how sperm acquire motility in the epididymis and how motility is altered in the female reproductive tract. Recent data show that the sperm specific protein phosphatase PP1γ2, glycogen synthase kinase 3 (GSK3), and the calcium regulated phosphatase calcineurin (PP2B), are involved in epididymal sperm maturation. The protein phosphatase PP1γ2 is present only in testis and sperm in mammals. PP1γ2 has a isoform-specific requirement for normal function of mammalian sperm. Sperm PP1γ2 is regulated by three proteins – inhibitor 2, inhibitor 3 and SDS22. Changes in phosphorylation of these three inhibitors and their binding to PP1γ2 are involved in initiation and activation of sperm motility. The inhibitors are phosphorylated by protein kinases, one of which is GSK3. The isoform GSK3α is essential for epididymal sperm maturation and fertility. Calcium levels dramatically decrease during sperm maturation and initiation of motility suggesting that the calcium activated sperm phosphatase (PP2B) activity also decreases. Loss of PP2B results in male infertility due to impaired sperm maturation in the epididymis. Thus the three signaling enzymes PP1γ2, GSK3, and PP2B along with the documented PKA (protein kinase A) have key roles in sperm maturation and hyperactivation. Significantly, all these four signaling enzymes are present as specific isoforms only in placental mammals, a testimony to their essential roles in the unique aspects of sperm function in mammals. These findings should lead to a better biochemical understanding of the basis of male infertility and should lead to novel approaches to a male contraception and managed reproduction. Testicular spermatozoa in mammals are immotile and lack the ability to fertilize eggs. Motility initiation and fertilizing ability develop during their passage through the epididymis . EpididySperm flagellar activity and also ciliary motility of somatic cells are known to be regulated by the intracellular mediators cAMP, calcium and pH. Capacitation is operationaly defined as functional modifications that enable sperm to fertilize eggs. Sperm undergo various changes during capacitation: (a) removal of membrane cholesterol followed leading to a decrease in cholesterol to phospholipid ratio (b) membrane hyperpolarization, rise in intra-sperm pH and cAMP levels, (c) increase in calcium uptake, and (d) an increase in protein tyrosine phosphorylation possibily due to increased protein kinase A (PKA) activity . It is gConsiderable progress has been made in understanding changes in sperm cAMP, calcium, and pH during sperm activation and hyperactivation. The proteins responsible for the generation of cAMP, calcium influx and changes in pHi (intracellular pH) have been identified and their functions elucidated by biochemical and genetic approaches. Despite this information, we still do not understand, in biochemical terms, how these second messengers enable sperm to acquire motility in the epididymis and how motility is altered in the female reproductive tract. Our understanding is incomplete because other signaling enzymes can profoundly alter the action and effects of second messengers in sperm. These signaling enzymes, protein phosphatase 1 (PP1), glycogen synthase kinase 3 (GSK3) and the calcium activated phosphatase (PP2B), affect sperm function in the epididymis and in the female reproductive tract. Several reviews detail the roles of sperm cAMP-PKA and calcium metabolism . FollowiMotility can be induced in demembranated testicular and caput epididymal sperm in the presence of ATP and cAMP, as well as appropriate calcium levels and pH . Early sIt is well known that cAMP acts through a protein kinase (PKA). Knockout of the sperm sAC or PKA leads to infertility due to impaired sperm motility and the inability of sperm to undergo hyperactivation . Mature One of the downstream effects of PKA has been suggested to involve activation of protein tyrosine kinases (PTKs): proline−rich tyrosine kinase 2 (PYK2), ABL , SRC, and FER . Howeverin vitro, results in initiation of vigorous motility. More recently, elevation of pHi has also been recognized to be a key event required for initiation of calcium influx during sperm hyperactivation. Knockout of channels, Slo3, NHE1 (Slca9c) or SLC26A3, responsible for changes in pH renders sperm infertile  can be functionally replaced by one of the four mammalian PP1 isoforms, highlighting their functional equivalence . Becauseididymis . The enzididymis . Due to Ppp1cc is male infertility. Females lacking Ppp1cc are normal and fertile . Two other GS kinases, GSK-4 and -5, named based on their relative elution profiles in phospho-cellulose chromatography of muscle extracts, were later renamed as casein kinase 1 and 2, respectively . The enzGsk3β in mice causes late embryonic lethality  is a serine/threonine phosphatase regulated by calcium. In response to an elevation of cellular calcium, calmodulin binds to a calmodulin binding region of the catalytic subunit PPP3C. This binding causes an auto-inhibitory arm of calcineurin to move away from the substrate binding site thus activating the enzyme by enabling its access to substrates . The catertility . Sperm nertility .The questions of how calcineurin may act and how it is activated during epididymal sperm maturation were not addressed. Recent data show that calcineurin and GSK3 are interrelated in their roles in epididymal sperm maturation and absence of calcineurin increases GSK3 phosphorylation resulting in its lower catalytic activity . It is sAs described earlier the requirement and the roles of cAMP and the kinase activated by it, PKA, in sperm are well known. The relationship between PKA and GSK3 in sperm was indicated by the fact that cAMP analogs increased GSK3-α/β phosphorylation, without any isoform specificity. Phosphorylation of both GSK3 α and β isoforms were reduced and its catalytic activity increased in sperm with diminished cAMP , or due to knock out of sAC. That is, GSK3 is a target of PKA phosphorylation. This relationship between GSK3 and PKA was further validated in GSK3α knockout mice. Loss of GSK3α or β by targeted disruption or pharmacological inhibition of the enzyme significantly reduced sperm cAMP levels . The decInhibition of the predominant sperm protein phosphatase PP1γ2 by calyculin A significantly increased phosphorylation of both GSK3 isoforms (Ser21/9) in caudal epididymal sperm and a concomitant decrease in its catalytic activity. Conversely, increased GSK3 activity is associated with increased PP1γ2 activity. One of the ways by which GSK3 regulates PP1 activity is by its phosphorylation of the inhibitor I2 as discussed earlier. Phosphorylated I2 dissociates from PP1 leading to its activation . The rol2+ during hyperactivation is responsible for decreased phosphorylation and increased catalytic activity of GSK3α. Pharmacologic inhibition of calcineurin during capacitation abrogated this decrease in phosphorylation and the increase in GSK3 activity. Increased activities of GSK3α and PP2B appear to be characteristics of not only capacitated sperm, but also, paradoxically, of immature caput epididymal sperm. Thus, phosphatases (PP1γ2 and PP2B) and the kinases (PKA and GSK3) are mechanistically interrelated during epididymal initiation of motility and also during fertilization of the egg  with a unique six amino acid N-terminus due to expression of an alternate exon, exon 1b  Figure . The exoA germ cell–enriched protein, viz. sperm PKA interacting factor (SPIF), was found to be co-expressed and co-regulated with PKACα2 and with t-complex protein (TCP)-11 . These tThe GSK3α isoform is essential in mammalian sperm despite the fact both GSK3α and β isoforms are interchangeable in other cells and tissues. GSK3α arose in vertebrates presumably by gene duplication of GSK3β which is the only isoform in invertebrates. Among vertebrates the GSK3α isoform is absent in birds . SequencA relatively recent addition to the list of signaling enzymes regulating sperm function is the calcium regulated phosphatase, PP2B. Catalytic activity of PP2B is required in sperm during their passage through the epididymis as is the case with GSK3α. Sperm and testis express specific isoforms of the catalytic and regulatory calcineurin, PPP3CC and PPP3R2, respectively. Examination of the genomic sequences of several species shows that PPP3R2 is present only in eutherian mammalians and its amino acid sequence is remarkably conserved in the 121 mammals for which annotated genomic databases exist .Several predominantly testis-expressed proteins present in mammalian sperm have been identified as mammal-specific. These include proteins involved in DNA binding, sperm egg binding or those required for required for the unique structural features of mammalian sperm, such as protamine 3, SMCP, and ADAM proteins . HoweverIn summary, considerable data show that the enzymes PP1γ2, GSK3 and PP2B, along with PKA, are mechanistically interrelated in regulating the two physiological processes unique to mammals: epididymal sperm maturation and sperm hyperactivation preceding fertilization .SV and SD contributed to the conception and outline of the review. SD and CB organized the database. SV wrote the first draft of the manuscript. SD wrote the sections of the manuscript. All authors contributed to the manuscript reading and revisions.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.


Characterizing the phospho-proteome before and after selective inhibition of CDK12 activity by the analog 1-NM-PP1, we identified 5,644 distinct phospho-peptides, among which were 50 whose average relative amount decreased more than 2-fold after 30 min of inhibition (none of these derived from RPB1). Half of the phospho-peptides actually showed >3-fold decreases, and a dozen showed decreases of 5-fold or more. As might be expected, the 40 proteins that gave rise to the 50 affected phospho-peptides mostly function in processes that have been linked to CDK12, such as transcription and RNA processing. However, the results also suggest roles for CDK12 in other events, notably mRNA nuclear export, cell differentiation and mitosis. While a number of the more-affected sites resemble the CTD in amino acid sequence and are likely direct CDK12 substrates, other highly-affected sites are not CTD-like, and their decreased phosphorylation may be a secondary (downstream) effect of CDK12 inhibition.We asked whether the C-terminal repeat domain (CTD) kinase, CDK12/CyclinK, phosphorylates substrates in addition to the CTD of RPB1, using our CDK12 Human CDK12, a tumor suppressor for ovarian and other cancers ,4. CDK12By RNA-mediated knock-down or by chemical inhibition of catalytic activity, metazoan CDK12 has been implicated in several processes in vivo, and as anticipated these overlap with Ctk1-influenced processes in yeast. For example, mammalian CDK12 has been connected with expression of genes involved in DNA damage responses ,19, it hOverall, a picture is emerging in which CDK12 plays major roles in transcription, RNA processing and genome stability. Consistent with a role in genome stability, CDK12 was identified as a likely tumor suppressor for ovarian cancer several years ago ,26. Of mPhenotypes caused by knocking-down or inhibiting the activity of CDK12 are usually attributed to changes in phosphorylation of the CTD, but it is possible that this enzyme also phosphorylates other protein substrates and that altered phosphorylation of these non-CTD substrates leads to some of the observed phenotypes. Along these lines, other CTD kinases have been shown to perform functionally meaningful phosphorylation of non-CTD substrates. Thus, CDK9 phosphorylates SPT5, facilitating transformation of a paused pol II complex into an actively elongating one . In factanalog-sensitive HeLa cell line represented a good system for finding such substrates. This cell line (HeLa “CDK12as”) expresses only an analog-sensitive, selectively-inhibitable version of CDK12 n) would seem the most likely to be directly phosphorylated by CDK12, which prefers to add a phosphoryl group to the S2 or S5 position ; thus, CDK12 and its relatives are said to be ‘proline-directed’ kinases. Among the peptide sequences in As just mentioned, affected phosphorylation sites that resemble the CTD consensus sequence . Note tbottom). With this set of sequences we used <weblogo.berkeley.edu> to find a sequence logo potentially representing a CDK12 consensus phosphorylation site. The resulting logo B is of cper se. Perhaps the most likely CDK12 substrate is TPR, a component of the “basket” of the nuclear pore complex (NPC) , that coAnother likely substrate is LIMD1, which contains two CTD-similar phosphorylation sites showing reductions after CDK12 inhibition of ~5× and 3×, respectively. The most-affected site matches the sequence logo quite well [Px(S)PxVxxP]. The LIMD1 protein functions in a number of processes, including transcriptional repression of several signaling pathways and formation of P-bodies. The repertoire of direct CDK12-catalyzed phosphorylations will interestingly increase if LIMD1 is a true substrate. The peptide from ADAM17 decreases 5.3-fold, and it shows a good match to the sequence logo, except that it has a less-favored Arginine 3 positions C-terminal to the phosphorylated T residue [PxTPxRxxP]. The other peptides in WT cells were treated for 30 min with DMSO alone or with 1-NM-PP1 in DMSO, then cells were collected and extracts prepared as before. A phosphor-proteomics analysis of the CDK12WT samples identified 6,295 total P-peptides corresponding to 2201 phosphoproteins  were defined as those whose abundance changed >2-fold  in the presence of the analog. There were 68 P-peptides that decreased >2-fold after 1-NM-PP1 addition and 3 that increased >2-fold . Toward this end, we repeated the experiment of proteins , numbers >2-fold , again nas experiment also decreased in the CDK12WT experiment, we looked for P-peptides that changed >2-fold in both experiments; we found 6 in-common P-peptides that decreased and 1 in-common P-peptide that increased . Perhaps the most likely direct substrate is the protein TPR (ortholog of budding yeast Mlp1), which contains three CTD-similar sites at which phosphorylation decreases rapidly after inhibition of CDK12 activity . TPR yM functionUnexpectedly, the phospho-peptide that decreased the most after CDK12 inhibition (>12-fold) was from the DNA damage repair protein XPC. While the two affected phosphorylation sites in this XPC peptide are extremely different from the consensus CTD repeats see , it willAmong previously identified CDK12-associating proteins were some that interact with the 5′ cap-end of RNAPII-synthesized RNAs, and in an earlier paragraph we focused on the nuclear roles of these proteins, including mRNA nuclear export. On the other hand, these multi-functional proteins are perhaps better known for their roles in translation. Intriguingly, recent results from Jones and colleagues also connect CDK12 with proteins that bind the cap-end of RNAPII transcripts (notably the cap-binding repressor 4E-BP1), and in this case the result of CDK12 activity is in fact regulation of translation . After Cas-inhibitory analog, suggesting an important in vivo role for this phosphorylation event.Another likely substrate of CDK12 is LIMD1, with two CTD-similar phosphorylation sites significantly affected by CDK12 inhibition . LIMD1 iOther proteins in the signaling/Txn-regulation group also show significant phosphate loss after CDK12 inhibition, but several of the sites involved do not resemble the CTD at all, such as those in MK03 and MK01 (known to be phosphorylated by MEK1/2), RGH35, ANS1A, KANK2 and BCAR3 cf. . We propSTSDIEEK) does not obviously resemble the CTD repeats. Another is NELFE, a subunit of transcription elongation factor NELF; again, however, the P-site (underlined) in NELFE (SISADDDLQESSR) is not CTD-like.Two other proteins, both with P-peptides showing a modest 2.2-fold decrease in amount after CDK12 inhibition , are worWT . This ein vitro ,41. InteWe previously showed that inhibiting CDK12 activity in living cells leads rapidly to altered phosphorylation states of the RNAPII CTD, and we now demonstrate that CDK12 inhibition also alters phosphorylation states of several dozen other proteins. From inspecting the amino acid sequences of the affected phosphorylation sites, we conclude that some are phosphorylated directly by CDK12, while others are phosphorylated by different kinases and are affected indirectly by CDK12 inhibition; however, the normal phosphorylation levels at all of the sites depend on CDK12 catalytic activity. Instructively, a number of CDK12-dependent phosphorylation events affect proteins involved in pre-mRNA processing and mRNA nuclear export, including nuclear pore complex components; these observations lead us to propose that CDK12 plays a role in functionally bridging between RNA processing machineries and the nuclear pore complex. In addition, CKD12 inhibition leads to dramatic dephosphorylation at a site in the XPC protein. Since both CDK12 and XPC are required for maintenance of the undifferentiated state of mouse embryonic stem cells, we speculate that this double requirement involves the CDK12-dependent XPC phosphorylation. Among the other phosphorylation events that depend on CDK12 activity are several that affect proteins involved in mitosis, and we surmise that defective phosphorylation of these proteins in CDK12-deficient ovarian and prostate cells contributes to tumor development. We hope that future use of the CDK12as cell line will provide a better understanding of these and other CDK12-dependent events, elucidating specific in vivo functions of CDK12 and providing new insights into its role as a tumor suppressor.


IIbβ3 activation and α-granule secretion, (ii) synergistic enhancement of PAR-mediated activation of the small GTPase RAP1, a regulator of integrin activation and (iii) phosphorylation of the PI3K effector Akt. More importantly, the synergistic effect of TPO on phosphorylation of extracellular-regulated kinase (ERK1/2) and thromboxane (TxA2) synthesis was dependent on both p110β and p110γ. p110β inhibition/deletion, or inhibition of p110γ, resulted in a partial reduction, whereas inhibiting both p110β and p110γ completely prevented the synergistic effect of TPO on ERK1/2 phosphorylation and TxA2 synthesis. The latter was ablated by inhibition of MEK, but not p38, confirming a role for ERK1/2 in regulating TPO-mediated increases in TxA2 synthesis. In conclusion, the synergistic effect of TPO on RAP1 and integrin activation is largely mediated by p110β, whereas p110β and p110γ contribute to the effect of TPO on ERK1/2 phosphorylation and TxA2 formation.Thrombopoietin (TPO) enhances platelet activation through activation of the tyrosine kinase; JAK2 and the lipid kinase phosphatidylinositide 3-kinase (PI3K). The aim of our study was to identify the PI3K isoforms involved in mediating the effect of TPO on platelet function and elucidate the underlying mechanism. We found that p110β plays an essential role in TPO-mediated (i) priming of protease-activated receptor (PAR)-mediated integrin α This cytokine can therefore play a pro-thrombotic and potentially pathogenic role in clinical conditions where it is found to be elevated. These conditions include altered bone marrow haematopoiesis/failure5, coronary artery disease6, acute angina7, sepsis8, inflammatory bowel disease9, burns patients10 and smokers11. Furthermore, TPO mimetics are used in the clinic to treat primary immune thrombocytopenia. These mimetics could potentially alter/restore platelet function causing either beneficial or detrimental effects not associated with the increase in platelet count, however information on this is still scarce and conflicting12. We and others have previously demonstrated that the mechanism by which TPO can enhance platelet activation is dependent on the activation of both the non-receptor tyrosine kinase janus kinase 2 (JAK2) and the lipid kinase phosphatidylinositide 3-kinase (PI3K)13.The endogenous myeloproliferative leukaemia protein (c-MPL) agonist; thrombopoietin (TPO) is a cytokine primarily involved in regulating platelet production, but it can also act as a platelet primer by enhancing platelet activation and function15. The p110β isoform has been robustly demonstrated to play a dominant role in regulating platelet function through use of pharmacological agents and genetic models. The deletion of the p110β isoform or expression of a kinase dead form results in near ablation of both thrombin and convulxin-mediated production of PIP3 and in the phosphorylation of the PI3K effector Akt in response to thrombin, convulxin, collagen, adenosine diphosphate (ADP) and the TxA2 analogue U4661917. Furthermore, p110β has been demonstrated to support platelet function and thrombus formation after arterial injury but is not involved in regulating the haemostatic response16. In contrast, more minor roles have been reported for the other isoforms. The p110γ isoform appears to be primarily involved in regulating platelet response to ADP, with platelets deficient in p110γ having impaired aggregation in response to ADP and ablated ADP-mediated Akt phosphorylation19. The p110δ isoform plays a minor functional role in signalling downstream of GPVI and integrin αIIbβ320, whereas the p110α isoform has an unusual role in regulating the ability of platelet priming agents to enhance platelet function24.Agonist-mediated activation of PI3K leads to the production of the second messenger molecule phosphatidylinositol -trisphosphate (PIP3) and is well established in playing a vital role in supporting platelet activation and thrombus formation. The class I PI3Ks of which there are four catalytic isoforms; are responsible for the production of PIP3. All four of these isoforms are expressed in platelets with the p110β isoform being the most abundant and p110δ the least IIbβ3 activation and α-granule secretion is primarily mediated through the PI3K isoform p110β and involves the synergistic activation of the small GTPase RAP1. Furthermore, we have evidence that TPO/c-MPL in platelets can signal through the PI3K isoform p110γ; an isoform more commonly associated with being activated downstream of G-protein-coupled receptors (GPCR). Activation of this isoform in combination with p110β by TPO, is a requirement for the synergistic activation of the mitogen-activated protein kinase (MAPK) extracellular signal–regulated kinase (ERK1/2) and subsequent priming of TxA2 production.The aim of this study was to investigate which PI3K isoforms can contribute to the priming effect of TPO on platelet function using a combination of pharmacological inhibitors and genetic mouse models. Here we demonstrate that the priming effect of TPO on integrin αIIbβ3 activation and α-granule secretion in washed platelets pre-treated with TPO in the absence and presence of the pan-PI3K inhibitor wortmannin. TPO markedly enhanced PAR-mediated integrin αIIbβ3 activation, resulting in a left-shift of the concentration-response curve  and an increase in maximal integrin activation , but maximal P-selectin exposure was unaltered. Wortmannin treatment reduced maximal PAR-mediated integrin activation by ~40%, whereas α-granule secretion was minimally affected. Furthermore, the priming effects of TPO on both integrin αIIbβ3 activation and α-granule secretion were prevented by pre-treating platelets with wortmannin  integrin αion Fig. . TPO alsion Fig. . The effnin Fig. . These r15 in platelets and has been demonstrated to play an integral role in platelet activation27 and in supporting platelet priming in combination with the p110α isoform24. Pre-treatment of platelets with the p110β inhibitor TGX-221 had a similar effect as wortmannin on integrin αIIbβ3 activation and α-granule secretion  or α-granule secretion  or increase maximal integrin activation.The class IA PI3K isoforms  are known to be activated downstream of tyrosine-kinase associated receptors via the recruitment of p85–p110 complexes and relief of p85 inhibition on the p110 catalytic subunits. Of these isoforms p110β is the most abundantion Fig. . Maximalion Fig. . In the IIbβ3 activation  elicited by sub-maximal concentrations of the PAR-4 selective agonist; AYPGKF . Interestingly, functional responses in mouse platelets compared to human appeared to be more susceptible to enhancement by TPO. However, in support of our findings in human platelets, this enhancement was suppressed in platelets deficient in the p110β isoform Fig. . SimilarIIbβ3 activation and granule secretion30. Here we demonstrate that TPO mediates a synergistic enhancement in RAP1 activation. Stimulation of platelets with a sub-maximal concentration of SFLLRN induced RAP1 guanosine-5′-triphosphate (GTP) loading above basal whereas TPO alone did not ; demonstrating that TPO can enhance platelet function independently on TxA2 generation. However, previous studies have demonstrated that TPO can also enhance secondary aggregation and this is blocked by acetylsalicylic acid/aspirin3. We therefore hypothesised that in the absence of indomethacin, TPO-mediated enhancement of secondary platelet aggregation would be inhibited by PD184352. Indeed, when using sub-threshold concentrations of SFLLRN, TPO mediated two distinct waves of aggregation with the secondary but not primary wave being blocked by PD184352  e.g. STAT3 and STAT5α/β34. Another event that occurs is the tyrosine phosphorylation of the p85 regulatory subunit of PI3K and its association with Gab1. This phosphorylation of p85 relieves its inhibitory activity on the p110 catalytic subunits resulting in an increase in PI3K activity and the phosphorylation of downstream effectors such as Akt. It is this increase in PI3K activity that is a key driver in enhancing PAR-mediated integrin activation and α-granule by TPO, with a likely role for the small GTPase RAP1 in this process. More specifically, based on our findings with both pharmacological inhibitors and genetic models, it is an increase in the activity of the p110β isoform of PI3K that mediates this TPO effect. In keeping with this observation, the phosphorylation of the PI3K effector Akt is considerably reduced by either treating platelets with TGX-221 or by knocking out p110β. However, residual phosphorylation when stimulating with TPO alone or TPO and a PAR agonist does remain. This contrasts with our findings with the pan-PI3K inhibitor wortmannin, indicating that TPO signals through multiple PI3K isoforms. Our findings suggest that c-MPL activation signals to a second PI3K isoform; p110γ. The evidence for this is that i) the p110γ inhibitor reduces TPO-mediated Akt phosphorylation and ii) the combination of p110β and p110γ inhibition ablates TPO-mediated Akt phosphorylation. Furthermore, deletion of the p110α isoform and p110δ inhibitors did not alter TPO-mediated phosphorylation of Akt (data not shown). The finding that TPO can signal and promote functional responses via p110γ is intriguing and unexpected. Activation of this PI3K isoform is typically thought to occur downstream of GPCRs, via the Gβγ subunits, with general opinion suggesting that p110γ is essential for GPCR but not tyrosine-kinase associated receptor, mediated activation of PI3K36. However, our data clearly shows that TPO, which signals through the cytokine receptor c-MPL, can enhance ERK phosphorylation and TxA2 synthesis through PI3K p110γ. Interestingly, there have been other reports of p110γ playing an important functional role downstream of other tyrosine-kinase associated receptors. A study by Alcázar et al.37, demonstrated that p110γ was activated by T cell receptor (TCR) ligation and that deletion of p110γ resulted in impairments in TCR signalling and T cell activation. Furthermore, Schmid et al.36, demonstrated a role for p110γ in regulating tumour inflammation initiated by a range of ligands coupling to receptor tyrosine kinases; VEGF-A, toll like/interleukin-1 receptors; IL-1β and cytokine receptors; IL-6.This and previous studies have demonstrated that TPO alone is unable to induce platelet functional responses, however when used in combination with other platelet agonists it can enhance aggregation, integrin α40. However, in platelets TPO binding c-MPL doesn’t result in the phosphorylation of either ERK1/2 or p3843. This is despite platelets expressing the requisite upstream signalling molecules and TPO being able to stimulate the activation of RAS42. One explanation for this is that in platelets, RAS activation is not sufficient for ERK activation, as activation of PKC is required for the activation of ERK at the level of/upstream of MEK42. However, despite the inability of TPO to induce phosphorylation/activation of MAPKs, it can subtly enhance phosphorylation of p38 and induce a striking highly synergistic enhancement in ERK1/2 phosphorylation. With the ability of TPO to induce these enhancements being not only dependent on the PI3K isoform p110β but also p110γ.In addition to PI3K, TPO binding to c-MPL stimulates the activation of at least two MAPK pathways; ERK1/2 and p38. This is mediated by the recruitment and tyrosine phosphorylation of Grb2, SHC, and SOS, and GTP loading of the small GTPase RAS. In haematopoietic stem cells and megakaryocytes this results in promotion of proliferation and differentiation2; a prothrombotic agent which activates and amplifies platelet responses through the thromboxane receptors. TxA2 is generated by thromboxane synthase from prostaglandin H2 (PGH2) which is itself generated from arachidonic acid by the aspirin target; cyclooxygenase. Previous work attributes the MAPK family members in being able to regulate TxA2 production through their ability to phosphorylate and therefore increase the catalytic activity of cytosolic phospholipase A2 (cPLA2). This enzyme in turn will hydrolyse various phospholipids, into arachidonic acid. Our and previous findings clearly demonstrate that TPO can markedly enhance TxA2 and, in agreement with van Willigen et al.,31 we believe this is mediated by the enhanced activation of ERK1/2. This is based on our finding that the MEK1/2 inhibitor PD184352, which blocks ERK1/2 phosphorylation/activation but not p38 activity, ablates the synergistic component of TxA2 generation induced by TPO and SFLLRN. Furthermore, the p38 inhibitor; VX-702, which specifically blocked the phosphorylation of known p38 substrates, but not the phosphorylation of ERK1/2 or p38 itself, did not ablate the synergistic generation of TxA2.In human platelets, the MAPK family members have been implicated in regulating the production of TxA2 generation induced by the combination of SFLLRN and TPO was drastically reduced by pan-PI3K inhibition. This reduction was an ablation of the synergistic component of TxA2 generation, as SFLLRN and TPO-mediated TxA2 generation in the presence of wortmannin was almost identical to TxA2 generation mediated by SFLLRN alone. In a further mirroring of our results on ERK1/2 phosphorylation, both the p110β and p110γ inhibitors could reduce the synergistic component and a further reduction was observed with combined inhibition. These data highlight and strengthen the idea that TPO through the PI3K isoforms; p110β and p110γ can synergistically increase SFLLRN-mediated activation of ERK1/2 which in turn leads to an enhancement in TxA2 production. Furthermore, this enhancement in TxA2 can contribute to a pro-aggregatory response under conditions where cyclooxygenase is not directly inhibited.In support of our findings regarding ERK1/2 phosphorylation, TxA2 independent and dependent pathways  was from Bachem . Protease activated receptor 4 (PAR-4)-activating peptide (AYPGKF-NH2) was from PeptideSynthetics . Recombinant human TPO and wortmannin were from Bio-Techne . Recombinant murine thrombopoietin (TPO) was from PeproTech . TGX-221, AS-252424, PD184352, IC-87114, PIK-75 and VX-702 were from Selleckchem . Mouse anti-human PAC-1 conjugated to FITC and mouse anti-human CD62P (AK-4) conjugated to PE were from BD Biosciences . Rat anti-mouse JON/A conjugated to PE and rat anti-mouse CD62P (Wug.E9) conjugated to FITC were from emfret ANALYTICS . Phospho-ERK1/2T202/Y204 (#9101), phospho-AktS473 (#4060), phospho-JAK2Y1007/1008 (#3771), phospho-STAT5α/βY694 (#4322), phospho-p38T180/Y182 (#4511), phospho-MAPKAPK2T334 (#3007), phospho-HSP27S78(#2405), ERK1/2 (#9102), Akt (#2920), and p110β (#3011) antibodies were from Cell Signaling Technology . Talin (#SC7534), GAPDH (#SC25778) and RAP1 (#SC65) antibodies were from Santa Cruz Biotechnology . Odyssey blocking buffer (TBS) was from LI-COR Biosciences . Secondary antibodies were from Jackson ImmunoResearch . TxB2 ELISA kit was from Enzo Life Sciences . All other materials were from Sigma Aldrich .Protease activated receptor 1 (PAR-1)-activating peptide . Blood was obtained with approval from the local Research Ethics Committee of the University of Bristol from healthy drug-free volunteers, who gave full informed consent in accordance with the Declaration of Helsinki.2 inhalation, in accordance with Schedule 1 of the Animals (Scientific Procedures) Act (1986), and blood was drawn by cardiac puncture into a syringe containing 4% trisodium citrate . For transgenic experiments appropriate wild-type (WT) controls were used . p110α flox/flox:Pf4-Cre mice (C57BL/6) and p110β mice (mixed C57BL/6J x 129 Sv) have been previously described44.All animal studies were approved by the local research ethics committee at the University of Bristol, UK and mice were bred and maintained for this purpose under the UK Home Office licence PPL30/3445. Mice (8–24 weeks old) were sacrificed by rising CO45. Platelets were resuspended in HEPES–Tyrode’s buffer , supplemented with 0.1% glucose (w/v), 10 μm indomethacin and 0.02 U mL−1 apyrase.Washed human and mouse platelets were prepared as previously describedIIbβ3 and α-granule secretion were assessed by flow cytometry as previously described using a BD AccuriTM C6 plus 46. For human platelets a FITC-conjugated antibody directed against the high affinity form of integrin αIIbβ3 (PAC-1) and PE-conjugated antibody for the α-granule marker CD62P (AK-4) were used. For mouse platelets a PE-conjugated antibody directed against the high affinity form of integrin αIIbβ3 (JON/A) and a FITC-conjugated antibody for the α-granule marker CD62P (Wug.E9) were used. For platelet aggregation, platelets (2 × 108/mL in the absence of indomethacin) were pre-incubated with inhibitors/TPO as indicated before agonist-stimulated aggregation was monitored using a CHRONO-LOG® Model 700 aggregometer at 37 °C, with continuous stirring at 1000 rpm. Data were recorded using Aggrolink Version 8 software (CHRONO-LOG®).Activation of integrin α8/mL) were stimulated as indicated and lysed directly in 4X NuPAGE sample buffer containing 0.5 M dithiothreitol (DTT). Proteins were resolved by electrophoresis as previously described45 and transferred onto polyvinylidene fluoride (PVDF) membranes. For some experiments, gels and membranes were cut into two parts to be probed separately with different primary antibodies47. Proteins were visualized by near-infrared detection using a LI-COR® Odyssey imaging system (providing a wide linear dynamic range) unless indicated otherwise. LI-COR® Image Studio  was used to create final images, the software analysis options only affect how raw data pixels are mapped to the screen and does not alter the experimental data. LI-COR® Image Studio  was used to quantify bands. Bands were defined using the rectangle shape tool to obtain fluorescence values and median local background (intensity of pixels in a border around the shape) was automatically subtracted.Washed platelets  as previously described2 generation was assessed by measuring TxB2 in platelet supernatants using a commercially available colorimetric ELISA kit  as previously described48. Briefly, platelets (4 × 108/ml in the absence of indomethacin) were stimulated as indicated before addition of 5 mM ethylenediaminetetraacetic acid and 200 μM indomethacin. Platelets were pelleted  and supernatant collected for analysis.TxAData were analysed and fitted using GraphPad Prism 7 software. All data are presented as the mean ± s.e.m of at least three independent observations. Concentration-response curves were fitted with a four-parameter logistic equation.Supplementary Figures