TLRs expression in canine mammary gland neoplasms: a pathological and molecular study.

TLRs are a class of PRRs that play a vital role in innate immunity. TLRs are expressed on immune cells and mammary epithelial cells. They can promote tumor growth, angiogenesis, invasion, and viability signaling. The current study aimed to test the correlation between histologic types and grades of neoplasms and TLRs gene expression levels. Twenty-one tissue samples of canine mammary neoplasms were stained with H&E. Then, it evaluated histologic type and grade according to the methods of Goldschmidt et al. and Peña, respectively. We established real-time PCR quantification assays to measure the mRNA abundances of TLRs in normal and neoplastic mammary glands. Profile pattern of TLR 1, 2, 3, 4, 5, 6, and 9 genes expression in canine mammary glands performed in 21 samples of mammary gland neoplasms and three non-neoplastic mammary gland samples from normal dogs. TLR 3, 4, and 9 mRNA overexpression were detected. In addition, tubulopapillary carcinoma grade II, SCC grade III, and carcinoma mixed type grade II demonstrated the highest relative TLR-3, and 9 mRNA expression levels. Complex carcinoma grade I, ductal carcinoma grade II, and anaplastic carcinoma grade II showed the highest relative TLR4 mRNA expression level. Although histopathological characteristics of tumors, including histologic type, grade, and inflammation, influenced TLRs mRNA expression level, such correlation was insignificant (P > 0.05).

Congenital cerebral and cerebellar anomalies in relation to bovine viral diarrhoea virus and Akabane virus in newborn calves.

Congenital malformations occur sporadically in cattle; however, congenital structural and functional disorders of the nervous system are rather common in ruminants. Among the numerous causes of congenital nervous system defects, infectious agents are highlighted in this paper. Virus-induced congenital malformations are well known, among which those caused by bovine viral diarrhoea virus (BVDV), Akabane virus (AKAV), Schmallenberg virus (SBV), Bluetongue virus (BTV), and Aino virus (AV) are the most studied. In this study, we specify and categorise macroscopic and histopathological lesions in the brain of 42 newborn calves suffering from severe neurologic signs and diagnosed with BVDV and AKAV infection. Following a complete necropsy, specimens were collected from the brains to track the presence of BVDV, AKAV and SBV utilising reverse transcription polymerase chain reaction. Of the 42 examined calves, 21 were BVDV positive and 6 were AKAV positive, while 15 brains were negative for the studied agents. Regardless of the aetiology, cerebellar hypoplasia, hydranencephaly, hydrocephalus, porencephaly, and microencephaly were detected. Cerebellar hypoplasia was the most common lesion seen in both BVDV-positive and AKAV-positive cases. Virus-induced necrosis of the germinative cells of the external granular layer of cerebellum, as well as vascular damages, are believed to be the underlying causes of cerebellar hypoplasia. BVDV was the most important aetiological agent of such cases in this study.

SARS-CoV-2 in domestic cats (Felis catus) in the northwest of Iran: Evidence for SARS-CoV-2 circulating between human and cats.

This study aimed to investigate the prevalence of COVID-19 in domestic cats, focusing on the disease in the northwest of Iran and then showing the natural transmission of SARS-COV-2 circulating between domestic cats and humans. After receiving ethic codes from Tehran University of Medical Sciences (IR.TUMS.VCR.REC.1399.303) and confirmed by the Center of Communicable Diseases Control (CDC) of Iran, 124 domestic cats were collected from the homes and only one hospital of Meshkin -Shahr district from northwestern Iran where SARS-CoV-2 patients were hospitalized and quarantined during 2020. Samples were prepared from fluid materials of oropharynx and nasopharynx. All samples were tested by real-time PCR (RT-PCR) using specific genes N and ORF1ab in Pasteur Institute of Iran, and then partial sequence analyses of S gene were performed. All collected cats were kept in separated cages until SARS-COV-2 infection was confirmed with the RT-PCR. RT- PCR Ct values of 123 collected cats were ≥40; thus, all of them showed negative results, but one of the collected cats with close contact with its owner, whom confirmed SARS-CoV-2 showed positive results with gene N(Ct=30) and gene ORF1ab (Ct=32). Furthermore, the positive pet cat showed respiratory and gastro-intestinal clinical manifestations, and its owner was infected with SARS-CoV-2 two weeks ago. Cats are susceptible animals to SARS-CoV-2 infection. Epidemiological evidence showed that SARS-COV-2 is able to transmit to healthy cats due to having close contact with its owner as a reverse zoonosis.

Effects of COX inhibitors on responsiveness of the tracheal tract to acetylcholine and histamine and their relationship with LTC4 and PGE2 levels of bronchoalveolar lavage fluid in allergic Guinea pigs.

Introduction: Nonsteroidal anti-inflammatory drugs (NSAIDs) intervene in the COX (cyclooxygenase) pathways which generate two important inflammation mediators, prostaglandins (PGs) and leukotriene (LTs). Contradictory claims regarding the effect of NSAIDs in asthmatic patients continues to be an issue. The present study investigated the effects of COX inhibitors on the responsiveness of the tracheal tract and on the levels of LTC4 and PGE2 in cells of the bronchoalveolar lavage fluid in an allergic guinea pig model.Materials and Methods: Adult male Dunkin-Hartley guinea pigs (250 - 300 g) were divided into seven groups of six animals each. Four COX inhibitors, aspirin (200 mg/kg and 20 mg/kg), indomethacin (10 mg/kg), ketoprofen (10 mg/kg), and celecoxib (25 mg/kg), were given orally on day 17 to allergy induced guinea pigs at 0, 12, and 24 h before ovalbumin challenge on day 18. PGF2 and LT4 were measured in the bronchoalveolar lavage fluid as well as inflammatory cell count and total protein. Tracheal responsiveness to acetylcholine (Ach) and histamine (His) also was evaluated.Results: An augment in the response of the trachea to Ach and His, as well as overt allergenic signs including short breath, wheezing and sneezing, was observed. The most significant increase in tracheal hyper-responsiveness was observed in the ketoprofen-treated group with similar but less pronounced changes observed in the indomethacin-treated group. Although some variables increased with the aspirin and celecoxib treatments, overall the tracheal sensitivity was reduced. Inflammatory cells including eosinophils and neutrophils corresponded to the changes observed for each treatment group.Conclusion: Ketoprofen and indomethacin increased the tracheal sensibility to Ach and His; therefore, their administration is not recommended in patients susceptible to allergy.

Author Correction: Opium Consumption and the Incidence of Cancer: Does Opium Account as an Emerging Risk Factor for Gastrointestinal Cancer?

The original version of this article unfortunately contained a mistake in the author group section. The correct name of the fourth author is "Reza Shirkoohi."The original article has been corrected.

Opium Consumption and the Incidence of Cancer: Does Opium Account as an Emerging Risk Factor for Gastrointestinal Cancer?

PURPOSE: Some epidemiological studies have shown an association between opium consumption and the incidence of gastrointestinal (GI) cancer. The present study was designed to investigate the effects of opium on the initiation of GI cancer in rats. METHODS: Forty-five rats were randomly divided into three groups; each received different treatment for 40 weeks. The rats in group 1 received purified water, while animals in group 2 were treated with 5 mg/kg diethylnitrosamine (DEN) orally for 8 weeks and continued with purified water by the end of the experiment. The third experimental group received 300 mg/kg opium for 16 weeks and then continued with 50 mg/kg phenobarbital by the end of the 40th week. The growth of tumors in the treated groups was assessed by histological changes and the up/down expression of p53, cdkn1, cdk2, e-cdh, and n-cdh genes in different parts of GI tract. RESULTS: Histological examinations revealed that DEN was able to induce the growth of tumor in GI tract as shown by active mitotic figure in different regions of GI system and hyperplasia of hepatocytes associated with infiltration of inflammatory cells, intestinal villous hypertrophy, and colorectal adenoma. There was also significant (p < 0.05) overexpression of p53, cdk2, and n-Cdh genes in different parts of digestive system in DEN-treated group. However, these pathological changes and the degradation of gene expression were not observed in the opium-treated group. CONCLUSION: The results of this study suggest that the opium does not promote the initiation of cancer in GI tract.

The Effect of Oliveria Decumbens and Pelargonium Graveolens on Healing of Infected Skin Wounds in Mice.

BACKGROUND: Staphylococcus aureus is one of the most causative organisms in the skin wound infections. Development of resistant S. aureus to current treatments in individuals with low immunity is a global concern. The aim of this study was to evaluate the efficacy of herbal formulation against skin wound infection. METHODS: The efficacy of herbal formulation containing Oliveria decumbens and Pelargonium graveolens essential oils was evaluated in comparison to mupirocin against Methicillin Resistant S. aureus (MRSA) related skin wound infection in mice animal model. RESULTS: The herbal cream and mupirocin decreased the log CFU by 2.5±0.26 and 2.46±0.32, respectively, while the log CFU of S. aureus from wound skin were 5.9±0.26 and 5.65±0.23 for placebo and control groups, respectively. Moreover, the histological examinations showed that this cream improved the wound healing and increased the collagen deposition and wound contraction. CONCLUSION: This natural new formulation with O. decumbens and P. graveolens essential oils could be recommended as a new candidate for wound healing.

Fructooligosaccharide raftilose reduces the mycophenolate mofetil-induced complications: Hematological and biochemical alterations.

Mycophenolate mofetil (MMF) is a selective inhibitor of Inosine-5'-monophosphate dehydrogenase. Gastrointestinal (GI) disturbances in immature ones are reported for MMF-induced compilations, which in the case of occurrence dose reduction is required. Thus, in the present study, the fructooligosaccharide raftilose(®) (RFT) was co-administrated with MMF to estimate the protective effect of RFT against MMF-induced GI complications. Thirty six immature male Wistar rats were divided into six groups including: Control (normal saline), RFT-treated (100 mg kg(-1)), MMF-treated (20 mg kg(-1)), MMF + LRFT (50 mg kg(-1)), MMF + MRFT (100 mg kg(-1)) and MMF + HRFT (200 mg kg(-1)) groups. The hematocrit (Hct), lymphocyte/total WBC, feces water content and pH were analyzed. Moreover, the hepatic functional tests, kidney-related biomarkers, lipid and protein profiles, total antioxidant capacity (TAC), malondialdehyde (MDA) and nitric oxide (NO) contents were assessed. Co-administration of RFT stabilized the MMF-reduced body weight. The MMF significantly diminished Hct and lymph/total WBC (p < 0.05). Only MRFT enhanced the lymphocyte/total WBC. Increased water content, no changes in feces pH, increased serum ALT and AST, no alteration in urea and mild enhancement in creatinine were demonstrated in MMF-received animals. However, RFT at low dose ameliorated the feces parameters and reduced ALT. No significant changes were demonstrated for serum lipid and protein profiles in MMF- and RFT + MMF-treated groups. The RFT enhanced the serum TAC, reduced MDA and NO contents. In conclusion, our data suggested that RFT could be considered as an effective agent to subsidize the MMF-induced clinical, hematological and biochemical disorders.

Evaluation of Gastric Lesions Based on Helicobacter pylori and Helicobacter-Like Organisms (HLOs) in Cats; A Histopathological and Bacteriological Study.

BACKGROUND: The lesions induced by Helicobacter pylori in a candidate animal model should always be examined thoroughly. The resemblance of these lesions to those observed in humans can indicate whether the usage of this model will contribute to the understanding of the various pathogenic mechanisms involved in the development of human H. pylori-associated diseases. OBJECTIVES: The aim of this study was to perform a histopathological and bacteriological evaluation of gastric lesions based on H. pylori and Helicobacter-like organisms (HLOs) in cats. MATERIALS AND METHODS: The present study was carried out on 28 cat's (13 male and 15 female cases) gastric mucosae, which were tested by bacteriological and histopathological methods. Biochemical tests such as catalase, oxidase and urease were utilized in addition to Gram and Giemsa staining. RESULTS: This research demonstrated that solely one case of H. pylori was isolated by gastric mucosal culture. Microscopically, the infected stomachs by HLOs comprised a mild to severe diffuse lymphoplasmacytic infiltration into the subglandular and gastric mucosa. Lymphoid follicles were also marked, particularly within pyloric tissues and mostly in displaced mucosal glands. For 75% of the gastritis cases, both HLOs and rapid urease tests were positive, whereas 83% of cases were more than one-year-old with gastritis. Furthermore, 75% of cats indicated gastritis, though 25% encompassed no gastritis; hence 20% had negative results for the rapid urease test and 25% for the Giemsa staining test. Such results may indicate that cats without gastritis were considered as free of HLOs pathogenic bacteria. CONCLUSIONS: These results suggest that most cases of gastritis were located in the antral region. Additionally, the isolation of H. pylori from domestic cats raises the possibility of zoonotic characteristics for the slightly pathogen; therefore transmission occurs from cats to human and vice versa.

Detection of Helicobacter spp. DNA in the colonic biopsies of stray dogs: molecular and histopathological investigations.

BACKGROUND: In dogs, the gastric Helicobacter spp. have been well studied, but there is little information regarding the other parts of the alimentary system. The incidence of Helicobacter spp. infection in dogs is largely unknown and to our knowledge there are no data about their potential pathogenic role. In light of these considerations, the aims of this study were (i) to assess the prevalence of Helicobacter spp. in colonic biopsies of healthy and symptomatic stray dogs also (ii) we isolate and characterize helicobacters in canine colonic biopsies to compare the commonly used tests for the identification of Helicobacter spp. and to determine the occurrence of these species in dogs. METHODS: Tissues from fifteen stray dogs (8 males and 7 females, age 6 months -10 years) were used in this study. From each stray dog, multiple colonic biopsies were taken for PCR. Biopsies for PCR of cecum and colon were immediately frozen and stored at -20°C until DNA extraction. Samples for histological examination were fixed in 10% neutral buffered formalin and embedded in paraffin wax. RESULTS: In the cecum and colon, Helicobacter spp. DNA was detected in all dogs. H.canis, H.bizzozeronii, H. bilis, H.felis, H.salomonis and H.pylori Identified by specific polymerase chain reaction. Histopathology demonstrated that Helicobacter organisms were localized within the surface mucus and the intestinal crypts. Dogs with heavy Helicobacter spp. colonization were significantly in younger as well as had a higher level of mucosal fibrosis/atrophy than dogs with uncolonized or poorly colonized biopsies (p<0.05). CONCLUSIONS: We have indicated that the crypts of the cecum and colon of healthy and symptomatic dogs are heavily colonized by Helicobacter spp.. Combined molecular and histological approaches demonstrated that enterohepatic Helicobacter spp. infection is rather common in colonic biopsies of healthy and symptomatic stray dogs, with Helicobacter spp. specialy H. canis, H.bizzozeroni, H.billis, H.felis and H. salomonis identified as the most common species. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1957989294118782.

An overall assessment of circumanal gland adenoma in a terrier mix breed dog.

In September 2012, a 10-year-old, intact male, terrier mix breed dog was evaluated because of multiple, 0.5 to 1.2 cm in diameter, round, intradermal nodules around the anus. It had surgery to excise a firm, painful swelling in the left ventrolateral perianal region and the excision part was observed under light microscopy. The mass spreading in to sub acute was of left hind leg out from the ventro-lateral of anus, 1.2 cm×1 cm/ 0 cm×0.5 cm in size and 125 g in weight. A complete blood cell count, serum biochemistry panel, and urinalysis (cystocentesis sample) were evaluated. Significant laboratory data demonstrated microcytic anemia (hemoglobin of 6.4 mg/dL) and normal coagulation times. No remarkable abnormalities were found in the complete blood count and an ionized calcium of 1.91 mmol/L (reference range, 1.1-1.3 mmol/L) was confirmed hypercalcemia. On cytologic and histopathologic examinations, evaluation of the aspirate revealed a prominent population of round-to-polygonal nucleated cells arranged as cohesive groups with isolated individual cells. A mild degree of anisocytosis and anisokaryosis was observed. In addition, smaller reserve type cells, with darker cytoplasm and a higher nucleocytoplasmic ratio. The adenomas generally retain the lobular architecture, but some may contain focal areas of cellular pleomorphism. These changes may suggest malignant transformation and have led to discordant interpretations, the well-developed stroma surrounding the lobules and hepatoid cells was noted. Ulceration, hemorrhage, necrosis and secondary infection with inflammatory cell infiltrates are common. These cytology and histopathology features are consistent with hepatoid gland adenoma.

Cervical type AB thymoma (Mixed) tumour diagnosis in a mynah as a model to study human: clinicohistological, immunohistochemical and cytohistopathological study.

Thymoma is a primary mediastinal neoplasm arising from or exhibiting differentiation towards thymic epithelial cells, typically with the presence of non-neoplastic lymphocytes. A 13-year-old male Mynah bird (acridotheres tristis) was presented for evaluation of a 2.3 × 1.5 × 1.0 cm mass in the left ventrolateral cervical region. The clinical signs, radiology, cytohistopathology and immunohistochimy findings related to the thymoma are presented. These findings indicated that the tumor was a type AB thymoma according to the World Health Organization (WHO) and veterinary classification. Thymomas are rarely reported in avian species and this is the first report in a Mynah bird. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1159525819982779.

A report of left dorsal displacement of the large colon in a tropical horse.

We report one such case which was diagnosed intraoperatively as left dorsal colon volvulus due to multiple mesenteric abnormalities. A 17-year-old castrated male horse was taken to the Tehran University Veterinary Hospital for treatment of metacarpal wound accompanied by severe abdominal distension and acute colic. The treatment and measurement were taken for a month, and the prepared biopsy indicated that the healing trend was obvious. Unfortunately, prior to discharge, the clinical colic manifestations emerged and the animal suddenly died. Dilated large intestine was palpated per rectum and a ventral midline exploratory laparotomy was performed, a complete volvulus of the ascending colon was identified with multiple mesenteric anomalies of unknown aetiology. The pathologic changes observed in this study accurately reflect those changes reported in horse with naturally occurring colonic volvulus and can serve as a reference for subsequent studies on attenuating bowel injury. The present study results can be used to make a scientific assessment of prognosis in the pre-operative, operative, and post-operative management of horses with large colon volvulus.

Using tracheal segments for replacement of cervical oesophagus: an experimental study.

OBJECTIVES: Segmental resection and anastomosis of oesophageal lesions are not performed as a routine clinical practice because of complications and associated problems, whereas tracheal resection and anastomosis are a routine clinical practice. In this experimental study, we resected a segment of cervical oesophagus and replaced it with a tracheal segment. METHODS: In eight dogs (mixed races), weighing 20-30 kg, ageing 1-2 years, under general anaesthesia, through a cervical incision, 5 cm of cervical trachea was separated while preserving its attachments to surrounding fibroareolar tissues. Afterwards, 5 cm of the oesophagus was resected and replaced with a prepared segment of the trachea. Oral liquids were started at the first post-operative day; the animals were kept for 2 months and then euthanized. Quality of swallowing and voice were evaluated. After an autopsy, anastomoses were examined grossly and histopathologically. RESULTS: No complications occurred during surgery. Swallowing function and voice were normal in all eight dogs after the operation. No sign of aspiration was seen in clinical and radiographic examinations after starting oral diet. In autopsy examination, anastomoses were patent without narrowing or abnormal mucosal changes. Remarkable histopathological findings in replaced tracheal segments were squamous metaplasia, atrophy and degeneration of mucosal glands and degeneration of cartilages. CONCLUSIONS: Replacement of a segment of the oesophagus with an autogenous tracheal segment is a practical procedure with low complications and can probably be used for the treatment of cervical oesophageal lesions in human beings.

The efficacy and safety of long-term Helicobacter species quadruple therapy in asymptomatic cats with naturally acquired infection.

Owing to rising drug-resistant Helicobacter species infections in people and animals, currently therapies are losing their efficacy; therefore, regimens efficacious in the presence of drug resistance are needed. This study assessed the efficacy and safety of a 14-day quadruple Helicobacter species therapy in cats with naturally acquired infection. Thirteen asymptomatic adult stray cats with Helicobacter species infection (identified by analysis of gastric biopsies using polymerase chain reaction and Helicobacter-specific primers) received omeprazole 0.7mg/kg q 8h plus amoxicillin 20mg/kg q 12h, metronidazole 20mg/kg q 12h and clarithromycin 7.5mg/kg q 12h, for 14 days. Second molecular analysis of gastric biopsies revealed persistence of Helicobacter species DNA in four cats that were negative on quantitative urease testing, cytology and histopathology. Our results suggest that antibiotic regimens that are effective against Helicobacter pylori in people cannot eradicate Helicobacter species in cats with naturally acquired infection, although transient suppression may occur.

Histological and biochemical alterations in early-stage lobar ischemia-reperfusion in rat liver.

AIM: To investigate the structural and biochemical changes in the early stage of reperfusion in the rat livers exposed to lobar ischemia-reperfusion (IR). METHODS: The median and left lobes of the liver were subjected to 60 min ischemia followed by 5, 10, 30, 45, 60 and 120 min reperfusion. Blood samples were taken at different time intervals to test enzyme activities and biochemical alterations induced by reperfusion. At the end of each reperfusion period, the animals were killed by euthanasia and tissue samples were taken for histological examination and immunohistochemistry. RESULTS: Cell vacuolation, bleb formation and focal hepatitis were the most important changes occur during ischemia. While some changes including bleb formation were removed during reperfusion, other alterations including portal hepatitis, inflammation and the induction of apoptosis were seen during this stage. The occurrence of apoptosis, as demonstrated by apoptotic cells and bodies, was the most important histological change during reperfusion. The severity of apoptosis was dependent on the time of reperfusion, and by increasing the time of reperfusion, the numbers of apoptotic bodies was significantly enhanced. The amounts of lactate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, creatinine and urea were significantly increased in serum obtained from animals exposed to hepatic IR. CONCLUSION: Inflammation and subsequent apoptotic cell death were the most important changes in early-stage hepatic reperfusion injury, and the number of apoptotic bodies increased with time of reperfusion.

Assessment of chronic gastritis in pet dogs and its relation with helicobacter-like organisms.

The aim of this study was to evaluate the prevalence of chronic gastritis in pet dogs, to determine the histopathologic changes of gastric mucosa and, to determine its relationship with canine gastric Helicobacter infection. Sixty percent (n = 18), 27% (n = 8) and 13% (n = 4) of the examined stomachs showed normal, congested and erosive gastric mucosa respectively. Histopathologic examination was confirmed the presence of chronic gastritis in 40% of dogs (n = 12). Lymphocytic-plasmacytic gastritis was the most common type of chronic gastritis. Gastric Helicobacter was detected in cytological examination of 26 out of 30 dogs (86.6%) but in the PCR analysis, 93% of gastric samples were positive for GHLO. There was no significant relation between the presence of Helicobacters and chronic gastritis (p>0.05). Follicular gastritis was detected in 12 cases (40%) and there was also no significant correlation between its presence and GHLO's infection (p>0.05). In conclusion, chronic gastritis can be considered as a prevalent disease especially in dogs. Nutritional and environmental factors as well as individual immune response may have role in induction of chronic gastritis, but the clinical significance of these histopathologic changes should be evaluated.

Effects of vitamin D in lung, stomach, esophagus and testis tissues following administration of urethane in balb/c mice.

OBJECTIVE: To elucidate the role of vitamin D in the histopathological alterations process and K-ras gene mutation exon 1 of tissues including lung, stomach, esophagus, and testis, by the administration of urethane. METHODS: An experimental study in inbred balb/c mice aged 9-11 weeks was designed. This investigation was performed from 2003 to 2005 in the Department of Medical Genetics, Medical Sciences/University of Tehran, Tehran, Iran. The samples were classified into 3 groups: the urethane group was characterized by intraperitoneal injection of 3 times urethane (600 mg/kg/day at 48 hour intervals). The second group U+D was given 3.5 mg/kg (6.3 mg/1000 ml) vitamin D in the drinking water for 4 weeks following the same intake of urethane as the first group, and the third one was the control group. All mice were sacrificed after 20 weeks, tissues were removed and examined for histopathological changes and mutations in the exon 1 of the K-ras gene. RESULTS: Thirty mice were studied. The formation of lung tumor was, significantly, increased in the urethane group as compared with the control group (p<0.005), however, such a difference was not found in the U+D and control groups. In addition, there was no significant difference between all groups in other examined tissues. There was no mutation in the exon 1 of K-ras gene of the lung adenomas, adenocarcinomas, and stomach metaplasia. CONCLUSION: Our results showed the anti-tumorigenic effect of vitamin D3 in lung tumors induced by urethane. Vitamin D may reduce the risks of a tumorigenic diet that includes high fermented foods and beverages that produce urethane in their process.