Arul Prakash M

@tanuvas.org.in

Assistant Professor
TANUVAS



              

https://researchid.co/drarullpm

RESEARCH INTERESTS

Sensors in Livestock Production; Male reproductive management

29

Scopus Publications

433

Scholar Citations

11

Scholar h-index

14

Scholar i10-index

Scopus Publications

  • Cryopreservation process alters the expression of genes involved in pathways associated with the fertility of bull spermatozoa
    John Peter Ebenezer Samuel King, Manish Kumar Sinha, Arumugam Kumaresan, Pradeep Nag, Mohua Das Gupta, Mani Arul Prakash, Thirumala Rao Talluri, and Tirtha Kumar Datta

    Frontiers Media SA
    In bovines, cryopreserved semen is used for artificial insemination; however, the fertility of cryopreserved semen is far lower than that of fresh semen. Although cryopreservation alters sperm phenotypic characteristics, its effect on sperm molecular health is not thoroughly understood. The present study applied next-generation sequencing to investigate the effect of cryopreservation on the sperm transcriptomic composition of bull spermatozoa. While freshly ejaculated bull spermatozoa showed 14,280 transcripts, cryopreserved spermatozoa showed only 12,375 transcripts. Comparative analysis revealed that 241 genes were upregulated, 662 genes were downregulated, and 215 genes showed neutral expression in cryopreserved spermatozoa compared to fresh spermatozoa. Gene ontology analysis indicated that the dysregulated transcripts were involved in nucleic acid binding, transcription-specific activity, and protein kinase binding involving protein autophosphorylation, ventricular septum morphogenesis, and organ development. Moreover, the dysregulated genes in cryopreserved spermatozoa were involved in pathways associated with glycogen metabolism, MAPK signalling, embryonic organ morphogenesis, ectodermal placode formation, and regulation of protein auto-phosphorylation. These findings suggest that the cryopreservation process induced alterations in the abundance of sperm transcripts related to potential fertility-associated functions and pathways, which might partly explain the reduced fertility observed with cryopreserved bull spermatozoa.

  • Single nucleotide polymorphisms cumulating to genetic variation for fertility in crossbred (Bos taurus × Bos indicus) bull spermatozoa
    Manish Kumar Sinha, Arumugam Kumaresan, Thirumala Rao Talluri, John Peter Ebenezer Samuel King, Mani Arul Prakash, Pradeep Nag, Nilendu Paul, Kathan Raval, Elango Kamaraj, and Aranganathan V

    Informa UK Limited
    Spermatozoa from high-fertile (HF) and low-fertile (LF) breeding bulls were subjected to high-throughput next-generation sequencing to identify important Single nucleotide polymorphisms (SNPs) and novel variants associated with fertility. A total of 77,038 genome-wide SNPs were identified, among which, 10,788 were novel variants. A total of 42,290 and 34,748 variants were recorded with 6115 and 4673 novel variants in in HF and LF bulls, respectively. Higher number of SNPs were identified in HF compared to LF bulls. GO analysis of filtered genes with significant variations in HF bulls indicated their involvement in oxidative phosphorylation and metabolic pathways. GO analysis of filtered genes with significant variation in LF bulls revealed their involvement in Ca2++ ion binding, structural constituent of ribosome, and biological processes like translation and ribosomal small subunit assembly. The study identified SNPs in candidate genes including TPT1, BOLA-DRA, CD74, RPS17, RPS28, RPS29, RPL14, RPL13, and RPS27A, which are linked to sperm functionality, survival, oxidative stress, and bull fertility. The identified SNPs could be used in selection of bulls for high fertility and the variation in these genes could be established as an explanation for the fertility differences in bulls upon validation in large number of bulls.

  • Comparative Transcriptomic Analysis of Spermatozoa From High- and Low-Fertile Crossbred Bulls: Implications for Fertility Prediction
    Mani Arul Prakash, Arumugam Kumaresan, John Peter Ebenezer Samuel King, Pradeep Nag, Ankur Sharma, Manish Kumar Sinha, Elango Kamaraj, and Tirtha Kumar Datta

    Frontiers Media SA
    Crossbred bulls produced by crossing Bos taurus and Bos indicus suffer with high incidence of infertility/subfertility problems; however, the etiology remains poorly understood. The uncertain predictability and the inability of semen evaluation techniques to maintain constant correlation with fertility demand for alternate methods for bull fertility prediction. Therefore, in this study, the global differential gene expression between high- and low-fertile crossbred bull sperm was assessed using a high-throughput RNA sequencing technique with the aim to identify transcripts associated with crossbred bull fertility. Crossbred bull sperm contained transcripts for 13,563 genes, in which 2,093 were unique to high-fertile and 5,454 were unique to low-fertile bulls. After normalization of data, a total of 776 transcripts were detected, in which 84 and 168 transcripts were unique to high-fertile and low-fertile bulls, respectively. A total of 176 transcripts were upregulated (fold change > 1) and 209 were downregulated (<1) in low-fertile bulls. Gene ontology analysis identified that the sperm transcripts involved in the oxidative phosphorylation pathway and biological process such as multicellular organism development, spermatogenesis, and in utero embryonic development were downregulated in low-fertile crossbred bull sperm. Sperm transcripts upregulated and unique to low-fertile bulls were majorly involved in translation (biological process) and ribosomal pathway. With the use of RT-qPCR, selected sperm transcripts (n = 12) were validated in crossbred bulls (n = 12) with different fertility ratings and found that the transcriptional abundance of ZNF706, CRISP2, TNP2, and TNP1 genes was significantly (p < 0.05) lower in low-fertile bulls than high-fertile bulls and was positively (p < 0.05) correlated with conception rate. It is inferred that impaired oxidative phosphorylation could be the predominant reason for low fertility in crossbred bulls and that transcriptional abundance of ZNF706, CRISP2, TNP2, and TNP1 genes could serve as potential biomarkers for fertility in crossbred bulls.

  • Environmental factors affecting reproductive performance of Murrah buffaloes
    V. Boopathi, Shiv Prasad, A. Kumaresan, A. Manimaran, and M. Arul Prakash

    Informa UK Limited
    ABSTRACT Buffalo is the backbone of the farmer’s economy in Indian dairy industry. Data from 288 Murrah buffalo female calves maintained at the Livestock Research Center, Karnal, Haryana, born in the period between 2000 and 2009 were used for this study. Data were analysed using least-square procedures. Results indicated that season and period of birth had significant (P < 0.05) effect on reproductive performance of Murrah buffalo heifers. However, there was no significant effect of birth weight on different reproductive performance. The phenotypic parameter estimates indicated that the season and birth weight of a calf can be used as a selection tool for improvement of reproductive traits considering its high positive phenotypic correlations with succeeding reproductive traits.

  • RNA-Seq analysis reveals functionally relevant coding and non-coding RNAs in crossbred bull spermatozoa
    Mani Arul Prakash, Arumugam Kumaresan, Manish Kumar Sinha, Elango Kamaraj, Tushar Kumar Mohanty, Tirtha Kumar Datta, and Jane M. Morrell

    Elsevier BV

  • Evaluation of indirect diagnostic tests and PBMC expression of innate immune genes in subclinical mastitis in dairy cows


  • Sub-fertility in crossbred bulls: Deciphering testicular level transcriptomic alterations between zebu (Bos indicus) and crossbred (Bos taurus x Bos indicus) bulls
    Kamaraj Elango, Arumugam Kumaresan, Ankur Sharma, Pradeep Nag, Mani Arul Prakash, Manish Kumar Sinha, Ayyasamy Manimaran, Ebenezer Samuel King John Peter, Sakthivel Jeyakumar, Sellappan Selvaraju,et al.

    Springer Science and Business Media LLC
    Abstract Background The incidence of poor semen quality and sub-fertility/infertility is higher in crossbred as compared to Zebu males. Several attempts have been made to understand the possible reasons for higher incidence of fertility problems in crossbred males, at sperm phenotype, proteome and genome level but with variable results. Since the quality of the ejaculated spermatozoa is determined by the testicular environment, assessing the testicular transcriptome between these breeds would help in identifying the possible mechanisms associated with infertility in crossbred bulls. However, such information is not available. We performed global transcriptomic profiling of testicular tissue from crossbred and Zebu bulls using Agilent Bos taurus GXP 8X60k AMADID: 29411 array. To the best of our knowledge, this is the first study comparing the testicular mRNAs between crossbred and Zebu bulls. Results Out of the 14,419 transcripts detected in bovine testis, 1466 were differentially expressed between crossbred and Zebu bulls, in which 1038 were upregulated and 428 were downregulated in crossbred bulls. PI4KB and DPY19L2 genes, reported to be involved in sperm capacitation and acrosome formation respectively, were among the top 10 downregulated transcripts in crossbred testis. Genes involved in ubiquitination and proteolysis were upregulated, while genes involved in cell proliferation, stem cell differentiation, stem cell population maintenance, steroidogenesis, WNT signalling, protein localization to plasma membrane, endocannabinoid signalling, heparin binding, cAMP metabolism and GABA receptor activity were downregulated in crossbred testis. Among the 10 genes validated using qPCR, expression of CCNYL, SOX2, MSMB, SPATA7, TNP1, TNP2 and CRISP2 followed the same trend as observed in microarray analysis with SPATA7 being significantly downregulated and transition proteins (TNP1, TNP2) being significantly upregulated in crossbred bulls. Conclusions Abundant proteolysis by ubiquitination and downregulation of WNT signaling, cell proliferation, differentiation and steroidogenesis might be associated with higher incidence of poor semen quality and/or sub-fertility/infertility in crossbred bulls as compared to Zebu bulls. Downregulation of SPATA7 (Spermatogenesis Associated 7) and upregulation of transition proteins (TNP1 and TNP2) in crossbred bull testis might be associated with impaired spermatogenesis processes including improper chromatin compaction in crossbred bulls.

  • The proportion of tyrosine phosphorylated spermatozoa in cryopreserved semen is negatively related to crossbred bull fertility
    Kolanjiyappan Vignesh, Kailasam Murugavel, Dourey Antoine, Mani Arul Prakash, Kausthub Kishore Saraf, Pradeep Nag, Thirumalaisamy Karuthadurai, and Arumugam Kumaresan

    Elsevier BV

  • USE OF TAMARIND SEED COAT POWDER FOR CONTROLLING COCCIDIOSIS IN GOATS
    C. Soundararajan, K. Nagarajan, A.C. Satish, and M. Arul Prakash

    Diva Enterprises Private Limited

  • Occurrence of flea infestation on goats under stall fed condition and its control
    C. Soundararajan, K. Nagarajan, and M. Arul Prakash

    Springer Science and Business Media LLC

  • Behavioural adaptation of crossbred cows in automatic concentrate feeding station


  • Tick infestation on sheep, goat, horse and wild hare in Tamil Nadu
    C. Soundararajan, K. Nagarajan, S. Muthukrishnan, and M. Arul Prakash

    Springer Science and Business Media LLC

  • Tick infestation in human beings in the Nilgiris and Kancheepuram district of Tamil Nadu, India
    C. Soundararajan, K. Nagarajan, and M. Arul Prakash

    Springer Science and Business Media LLC

  • Effect of Boswellia serrata on hepatic biomarkers and cytokines in experimental rheumatoid arthritis


  • Distribution of larval and pupal stages of Simulium (Diptera: Simuliidae) flies in the Nilgiris hills of Tamil Nadu
    C. Soundararajan, K. Nagarajan, and M. Arul Prakash

    Springer Science and Business Media LLC

  • Pathological features of sheep pox seen in Madras red sheep in Tamil Nadu


  • Occurrence of aural myiasis in madras red sheep and its management by herbal medicine (Tamarind Seed Coat Powder)


  • Aural haematoma in a Jamunapari goat and its management


  • Sarcoptic mange infestation in rabbits in an organized farm at Tamil Nadu
    M. Arul Prakash, C. Soundararajan, K. Nagarajan, P. Tensingh Gnanaraj, and V. Ramesh Saravanakumar

    Springer Science and Business Media LLC

  • Peromelia in country chicken


  • Occurrence of polymastia in sheep


  • Monopodia in a country chicken - A first case report


  • Infrared thermography: A potential noninvasive tool to monitor udder health status in dairy cows
    M. Sathiyabarathi, S. Jeyakumar, A. Manimaran, G. Jayaprakash, Heartwin A. Pushpadass, M. Sivaram, K. P. Ramesha, D. N. Das, Mukund A. Kataktalware, M. Arul Prakash,et al.

    Veterinary World
    The animal husbandry and livestock sectors play a major role in the rural economy, especially for the small and marginal farmers. India has the largest livestock population in the world and ranks first in the milk production. Mastitis is the most common and expensive infectious disease in dairy cattle. The global economic losses per year due to mastitis amounts to USD 35 billion and for Indian dairy industry ₹6000 crores per year. Early detection of mastitis is very important to reduce the economic loss to the dairy farmers and dairy industry. Automated methods for early and reliable detection of mastitis are currently in focus under precision dairying. Skin surface temperature is an important indicator for the diagnosis of cow’s illnesses and for the estimation of their physiological status. Infrared thermography (IRT) is a simple, effective, on-site, and noninvasive method that detects surface heat, which is emitted as infrared radiation and generates pictorial images without causing radiation exposure. In human and bovine medicine, IRT is used as a diagnostic tool for assessment of normal and physiological status.

  • Prevalence of Rhipicephalus sanguineus on dogs in Chennai, Tamil Nadu


  • Indigenous ethnoveterinary medicinal practices for management of mastitis in dairy cattle
    P. Mooventhan, A. Manimaran, R. Senthil Kumar, A. Sakthivel Selvan, and M. Arul Prakash

    Agricultural Research Communication Center
    The role of ethno-veterinary medicine in livestock and human health is well known fact due to its widespread practice across the globe, including India. WHO stated that, 80% of people in developing countries depend on ethno-veterinary practices due to its inexpensive, easy accessibility and its preparations. Further, 50% of all modern drugs originally came from plants directly or its structural modification suggests for its potency and safety. Although, India has well practiced traditional knowledge, it could not capitalize the global market like USA, EU and China. Lack of well documentation of traditional “hidden” practices is one of the major limitations for its less exploitation. Therefore, an explorative study was conducted in participatory mode to explore and document the traditional practices for clinical mastitis management of dairy animals in the district of Bangalore urban, Karnataka. Since, the mastitis is most common and costly diseases of dairy animals in India and frequent reason for veterinary drug residue problem; in the present study, we have documented the indigenous practices and its procedure for the management of clinical mastitis in dairy animals.

RECENT SCHOLAR PUBLICATIONS

  • OPEN ACCESS EDITED BY
    A Mukherjee, T Luo, WS Kwon, A Kumaresan, JPES Kingt, MK Sinha, ...
    Application of genomics in livestock populations under selection or 2024

  • Single nucleotide polymorphisms cumulating to genetic variation for fertility in crossbred (Bos taurus Bos indicus) bull spermatozoa
    MK Sinha, A Kumaresan, T Rao Talluri, JP Ebenezer Samuel King, ...
    Animal Biotechnology 34 (7), 2875-2886 2023

  • Salivary crystallization pattern: a possible unconventional tool for timing of insemination and early pregnancy diagnosis in zebu cows
    NB Chavan, A Kumaresan, S Chhillar, S Nayak, MA Prakash, S Lathika, ...
    Journal of Dairy Research 90 (1), 21-25 2023

  • Study on mortality pattern of madras red sheep under field condition in Tamil Nadu
    PGDB T Chandrasekar, Pasupathi Karu, M Arul Prakash
    International Journal of Veterinary Sciences and Animal Husbandry 8 (6), 21-23 2023

  • RABBIT MANGE INFESTATION AND ITS THERAPEUTIC MANAGEMENT
    P Gopu, T Chandrasekar, MA Prakash, K Pasupathi, D Balasubramaniam
    Indian Journal of Veterinary and Animal Sciences Research 52 (2), 107-112 2023

  • UTILIZATION OF LIVESTOCK FARM SERVICES PROVIDED BY AGRICULTURAL TECHNOLOGY INFORMATION CENTRE (ATIC) OF TAMIL NADU VETERINARY AND ANIMAL SCIENCES UNIVERSITY
    MA Prakash, R Divisha
    Indian Journal of Veterinary and Animal Sciences Research 52 (3), 43-49 2023

  • Cryopreservation process alters the expression of genes involved in pathways associated with the fertility of bull spermatozoa
    JP Ebenezer Samuel King, MK Sinha, A Kumaresan, P Nag, M Das Gupta, ...
    Frontiers in Genetics 13, 1025004 2022

  • SNPs Cumulating to Genetic Variation for Fertility in Crossbred (Bos taurus X Bos indicus) Bull Spermatozoa
    MK Sinha, A Kumaresan, TR Talluri, JPES King, MA Prakash, P Nag, ...
    2022

  • Impact of lightning strike on economics of sheep and goat rearing in Tamil Nadu under field condition
    T Chandrasekar, P Karu, A Varun, D Balasubramanyam, MA Prakash
    Indian Journal of Animal Health 60 (1), 109-112 2021

  • Comparative transcriptomic analysis of spermatozoa from high-and low-fertile crossbred bulls: Implications for fertility prediction
    MA Prakash, A Kumaresan, JP Ebenezer Samuel King, P Nag, A Sharma, ...
    Frontiers in Cell and Developmental Biology 9, 647717 2021

  • Hermaphroditism in large white Yorkshire piglet: A case report
    M Arul Prakash, K Senthilkumar, D Balasubramanyam, T Chandrasekar
    2021

  • Successful management of rectal prolapsed in a large white Yorkshire – A case report
    DB S. Vidhya, M. Arul Prakash, K. Senthilkumar
    Indian Veterinary Journal 97 (10), 38-40 2021

  • Hermaphroditism in large white Yorkshire piglet: A case report
    MA Prakash, K Senthilkumar, D Balasubramanyam, T Chandrasekar
    The Pharma Innovation Journal 10 (7), 137-138 2021

  • Microtia in a Large White Yorkshire Piglet- A Case Report
    MA Prakash, K Sethilkumar, D Balasubramaniyam, C Jothika, ...
    International Journal of Livestock Research 11 (2), 190-193 2021

  • RNA-Seq analysis reveals functionally relevant coding and non-coding RNAs in crossbred bull spermatozoa
    MA Prakash, A Kumaresan, MK Sinha, E Kamaraj, TK Mohanty, TK Datta, ...
    Animal reproduction science 222, 106621 2020

  • Evaluation of indirect diagnostic tests and PBMC expression of innate immune genes in subclinical mastitis in dairy cows
    AC Hallolli, D Sharma, A Manimaran, A Kumaresan, M Sivaram, M Bagath, ...
    Indian Journal of Animal Sciences, 1103-1108 2020

  • Sub-fertility in crossbred bulls: deciphering testicular level transcriptomic alterations between zebu (Bos indicus) and crossbred (Bos taurus x Bos indicus) bulls
    K Elango, A Kumaresan, A Sharma, P Nag, MA Prakash, MK Sinha, ...
    BMC genomics 21 (1), 502 2020

  • The proportion of tyrosine phosphorylated spermatozoa in cryopreserved semen is negatively related to crossbred bull fertility
    K Vignesh, K Murugavel, D Antoine, MA Prakash, KK Saraf, P Nag, ...
    Theriogenology 149, 46-54 2020

  • இந்தியக் கோழி இனங்களும் சிறப்புப் பண்புகளும்
    MA Prakash, K Senthilkumar, D Balasubramaniam, C Soundararajan
    TANUVAS, Chennai 2020

  • இளம் பன்றிக் குட்டிகளைப் பராமரிக்கும் முறைகள்
    S Vidhya, K Senthilkumar, MA Prakash, T Balasubramaniam
    TANUVAS, Chennai 2020

MOST CITED SCHOLAR PUBLICATIONS

  • Infrared thermography: A potential noninvasive tool to monitor udder health status in dairy cows
    M Sathiyabarathi, S Jeyakumar, A Manimaran, G Jayaprakash, ...
    Veterinary world 9 (10), 1075 2016
    Citations: 77

  • Potential of acute phase proteins as predictor of postpartum uterine infections during transition period and its regulatory mechanism in dairy cattle
    A Manimaran, A Kumaresan, S Jeyakumar, TK Mohanty, V Sejian, ...
    Veterinary world 9 (1), 91 2016
    Citations: 47

  • Relationship of udder and teat conformation with intra-mammary infection in crossbred cows under hot-humid climate
    MRTKPJ Pranay Bharti, Champak Bhakat, Prabhat K. Pankaj, Showkat A. Bhat, M ...
    Veterinary world 8 (7), 898-901 2015
    Citations: 39

  • Comparative transcriptomic analysis of spermatozoa from high-and low-fertile crossbred bulls: Implications for fertility prediction
    MA Prakash, A Kumaresan, JP Ebenezer Samuel King, P Nag, A Sharma, ...
    Frontiers in Cell and Developmental Biology 9, 647717 2021
    Citations: 35

  • Sexing of spermatozoa in farm animals: a mini review.
    MA Prakash, AK Arumugam Kumaresan, AM Ayyasamy Manimaran, ...
    2014
    Citations: 26

  • RNA-Seq analysis reveals functionally relevant coding and non-coding RNAs in crossbred bull spermatozoa
    MA Prakash, A Kumaresan, MK Sinha, E Kamaraj, TK Mohanty, TK Datta, ...
    Animal reproduction science 222, 106621 2020
    Citations: 22

  • Sarcoptic mange infestation in rabbits in an organized farm at Tamil Nadu
    M Arul Prakash, C Soundararajan, K Nagarajan, P Tensingh Gnanaraj, ...
    Journal of parasitic diseases 41, 429-432 2017
    Citations: 21

  • Sub-fertility in crossbred bulls: deciphering testicular level transcriptomic alterations between zebu (Bos indicus) and crossbred (Bos taurus x Bos indicus) bulls
    K Elango, A Kumaresan, A Sharma, P Nag, MA Prakash, MK Sinha, ...
    BMC genomics 21 (1), 502 2020
    Citations: 18

  • Tick infestation on sheep, goat, horse and wild hare in Tamil Nadu
    C Soundararajan, K Nagarajan, S Muthukrishnan, M Arul Prakash
    Journal of parasitic diseases 42, 127-129 2018
    Citations: 18

  • Tick infestation in human beings in the Nilgiris and Kancheepuram district of Tamil Nadu, India
    C Soundararajan, K Nagarajan, M Arul Prakash
    Journal of parasitic diseases 42, 50-54 2018
    Citations: 14

  • Relationship of blood and seminal plasma testosterone concentrations with semen quality in crossbred bulls
    SLMAP S. K. Rajak, U. K. Tripathi, N. M. Attupuram, P. Boro, S. S. Layek, M ...
    Indian Journal of Dairy Science 67 (2), 162-167 2014
    Citations: 12

  • Effects of clinical mastitis on days open in dairy cattle and buffaloes
    A Manimaran, A Kumaresan, L Sreela, V Boopathi, M Arul Prakash
    Indian Vet. J 91 (12), 67-68 2014
    Citations: 11

  • Occurrence of flea infestation on goats under stall fed condition and its control
    C Soundararajan, K Nagarajan, MA Prakash
    Journal of parasitic diseases 42, 444-448 2018
    Citations: 10

  • Indigenous ethnoveterinary medicinal practices for management of mastitis in dairy cattle
    P Mooventhan, A Manimaran, RS Kumar, AS Selvan, MA Prakash
    Indian Journal of Animal Research 50 (1), 137-139 2016
    Citations: 10

  • The proportion of tyrosine phosphorylated spermatozoa in cryopreserved semen is negatively related to crossbred bull fertility
    K Vignesh, K Murugavel, D Antoine, MA Prakash, KK Saraf, P Nag, ...
    Theriogenology 149, 46-54 2020
    Citations: 9

  • Dairy farm labour utilization pattern and their welfare
    M Sathiyabarathi, S Jeyakumar, A Manimaran, G Jayaprakash, RD Kumar, ...
    Indian Journal of Dairy and Biosciences 26, 29-34 2015
    Citations: 7

  • Therapeutic management of concurrent sarcoptic and psoroptic mange infestation in rabbits
    R Divisha, C Soundararajan, MA Prakash
    J Entomol Zool Stud 8 (1), 1041-1043 2020
    Citations: 6

  • Automation in Feeding- A Precision Dairy Farm Management
    MSHAK M. Arul Prakash, T. Chandrasekar, R. Dinesh Kumar, A. Sakthivel Selvan ...
    Indian farmer 2 (5), 412-147 2015
    Citations: 5

  • Use of tamarind seed coat powder for controlling coccidiosis in goats
    ACSMAP C. Soundararajan, K. Nagarajan
    Indian Journal of Small Ruminants 25 (2), 247-250 2019
    Citations: 4

  • Environmental factors affecting reproductive performance of Murrah buffaloes
    V Boopathi, S Prasad, A Kumaresan, AMMA Prakash
    Biological Rhythm Research 2019
    Citations: 4