Assessing resuscitation in burn patients with varying degrees of liver disease.

We find minimal literature and lack of consensus among burn practitioners over how to resuscitate thermally injured patients with pre-existing liver disease. Our objective was to assess burn severity in patients with a previous history of liver disease. We attempted to stratify resuscitation therapy utilised, using it as an indicator of burn shock severity. We hypothesized that as severity of liver disease increased, more fluid therapy is needed. We retrospectively studied adult patients with a total body surface area (TBSA) of burn greater than or equal to 20% (n = 314). We determined the severity of liver disease by calculating admission Model for End-Stage Liver Disease (MELD) scores and measured resuscitation adequacy via urine output within the first 24 h. We performed stepwise, multivariable linear regression with backward selection to test our hypothesis with α = 0.05 defined a priori. After controlling for important confounders including age, TBSA, baseline serum albumin, total crystalloids, colloids, blood products, diuretics, and steroids given in first 24 h, we found a statistically significant reduction in urine output as MELD score increased (p < 0.000). In our study, severity of liver disease correlated with declining urine output during first 24-hour resuscitation more so than burn size or burn depth. While resuscitation is standardized for all patients, lack of urine output with increased liver disease suggests a new strategy is of benefit. This may involve investigation of alternate markers of adequacy of resuscitation, or developing modified resuscitation protocols for use in patients with liver disease. More investigation is necessary into how resuscitation protocols may best be modified.

Serial envenomation in a snake handler with severe coaguloapathy.

We present a rare case study of a 37-year-old man with a history of multiple prior snake bites who presented to the emergency department for treatment of a rattlesnake bite to his right hand. Upon examination, he was found to be mildly hypertensive and exhibited significant coagulation abnormalities. Initial treatment included six vials of Crotalidae polyvalent immune fab; however, his coagulopathy was so severe that he required an additional eight vials. Continuous monitoring and calculations of Snakebite Severity Score demonstrated resolution of coagulopathy within 36 hours of admission. We believe the patient's unusual recovery was likely due to an innate immune response, specifically an activated memory B-cell cascade. This case should lead researchers to consider that the resolution of severe coagulopathy might result from a memory-driven immune response in instances of multiple envenomations.

A Rare Mechanism of Inhalation Injury: Direct Thermal Damage to the Lower Airway.

In patients with inhalation injury associated with major burns, the primary mechanism of tissue harm depends on the location within the respiratory tract. Proximal to the trachea, the upper respiratory tract epithelium is classically injured via direct thermal injury. Such injury occurs due to the inhalation of high-temperature air. These upper airway structures and the tracheobronchial tree's dense vasculature protect the lower airways and lung parenchyma from direct thermal damage. The lower respiratory tract epithelium and lung parenchyma typically become injured secondary to the cytotoxic effects of chemical irritants inhaled in smoke as well as delayed inflammatory host responses. This paper documents a rare case in which a patient demonstrated evidence of direct thermal injury to the lower respiratory tract epithelium. A 26-year-old Caucasian male presented to the emergency room with 66% total body surface area thermal burns and grade 4 inhalation injury after a kitchen fire. Instead of visualizing carbonaceous deposits in the bronchi, a finding common in inhalation injury, initial bronchoscopy revealed bronchial mucosa carpeted with hundreds of bullae. Despite the maximum grade of inhalation injury per the abbreviated injury score and a 100% chance of mortality predicted with the revised Baux score, as well as a clinical course complicated by pneumonia development, bacteremia, and polymicrobial external wound infection, this patient survived. This dissonance between his expected and observed clinical outcome suggests that the applicability of current inhalation injury classification systems depends on the precise mechanism of injury to the respiratory tract. The flaws of these grading scales and prognostic indicators may be rooted in their failure to account for other pathophysiologic processes involved in inhalation injury. It may be necessary to develop new grading and prognostic systems for inhalation injury that acknowledge and better account for unusual pathophysiologic mechanisms of tissue damage.

Burn Management in a Patient With Acute Exacerbation of Comorbid Systemic Lupus Erythematosus.

A 28-year-old female presented to the burn unit with 2% total body surface area second-degree burns to the right flank and right breast after accidentally spilling coffee on herself while hospitalized for an acute exacerbation of systemic lupus erythematosus (SLE) in the form of neuromyelitis optica spectrum disorder. We document her inpatient management, which was challenging because of the contradictory relationship between typical management of SLE exacerbations (i.e., immunosuppressive medication regimens) and the body's post-burn healing process, which is inherently inflammatory in nature. Even with a high-dose immunosuppressive medication regimen, our patient's second-degree burns healed with non-operative management without significant adverse events. Adding to a small yet growing body of literature addressing the clinical presentation and management of burn wounds in the setting of an acute SLE exacerbation, our case suggests that clinicians must carefully weigh the risks of surgical intervention with those of non-operative management when approaching burn care during an acute rheumatologic disease flare up.

Acute Intermittent Porphyria in a Burn Patient: Case Study and Review of the Literature.

Healthcare providers evaluating patients presenting with neurological, visceral, or cutaneous symptoms that are disproportionate to the expected severity may need to consider porphyria in the differential. Porphyria is an inherited condition in which toxic metabolites of the heme pathway are increased. Carriers of porphyrias are asymptomatic and will not present with classical symptoms, nor will levels be elevated, until the disease is induced by certain drugs, hormones, or idiopathic causes such as the stress of trauma. Acute intermittent porphyria, a form of acute porphyria, is a rare autosomal dominant disease that results in a dysfunctional porphobilinogen deaminase. This consequently increases neurotoxic porphobilinogen and subsequent increase in δ-aminolevulinic acid. Both of these metabolites cause neurovisceral symptoms that afflict the patient in acute attacks. We present a rare case of acute intermittent porphyria manifested in a burn patient suffering a burn injury. The patient presented with symptoms indicative of acute intermittent porphyria, including altered mental status and abdominal pain accompanied with a chronic history of alcoholism and smoking. A negative work-up, including imaging and findings of associated manifestations consistent with Acute Intermittent Porphyria led to a discovery of elevated porphyrins. The patient's course and death due to his injuries gives insight into the presentation of acute intermittent porphyria in a burn patient.

Charge capture in the BICU: Increasing revenue by improving documentation behaviors.

Documentation by a healthcare provider is the key to capturing appropriate reimbursement for effort, expertize, and time given to patients. However, patient encounters are known to be under-coded; often describing a level of service that does not reflect the physician's labor. If there is deficient medical decision making (MDM) documentation, this will ultimately lead to a loss of revenue, as coders can only evaluate service levels from the documentation during the encounter. Physicians at the Timothy J. Harnar Regional Burn Center at Texas Tech University Health Sciences Center were experiencing below-average reimbursement for work performed in the burn center and theorized that deficiencies in documentation (particularly in the area of MDM) were the cause. They hypothesized that poor documentation by physicians was resulting in a substantial proportion of encounters being compulsorily coded at inadequate and imprecise levels of service. To improve the service levels of MDM in physician documentation at the Burn Center and consequently, increase the numbers and levels of billable encounters in the unit with an accompanying increase in revenue, two resources were created and employed with the purpose of providing increased documentation recall and thoroughness. These resources included a pocket card, designed to prevent missed details when documenting patient encounters, and a standardized EMR template that was mandated to be used by all BICU medical professionals rotating through the unit. After completion of the intervention period (July - October 2021), a comparison was made between the 4-month periods of July-October 2019 and 2021. Based on the encounters provided by residents and one fellow assigned to the BICU medical director, inpatient subsequent visit codes showed an average increase in billable encounters of 1500% amid the compared periods. Upon implementation of the intervention, subsequent visit codes 99231, 99232, and 99233 (higher-numbered codes indicating an increased level of service and accompanying reimbursement) raised by 142%, 2158%, and 2200%, respectively. An additional finding since the implementation of the pocket card and revised template, billable encounters have replaced the once-dominate global encounter, 99024 (associated with no reimbursement); realizing an increase in billable inpatient services due to complete and thorough documentation of non-global issues patients experienced throughout their hospital stays. Obtaining buy-in from physicians proved a significant challenge; consistent training and feedback allowed for an improved understanding of billing and coding processes within the BICU. The described findings indicate that a focused effort to improve documentation offers a promising method to yield potentially significant improvements in a unit's profitability.

Treatment of Severe Road Rash with ReCell® Autologous Skin Cell Suspension.

Road rash injuries are often variable in severity, with injuries ranging from simple scrapes to full-thickness burns. Autologous skin cell suspension devices, such as ReCell®, have shown increased promise by creating results comparable to the current standard of care, split-thickness skin grafting with significantly less donor skin required. We present a case of a 29-year-old male with significant road rash after a motorcycle accident at highway speeds, who was successfully treated solely with ReCell application. After surgery, he reported decreased pain with wound care and showed overall wound improvement with no changes in range of motion at 2-week follow-up. This case demonstrates the potential of ReCell as an independent treatment modality for pain and skin injury secondary to severe road rash.

Autologous Skin Cell Suspension in Toxic Epidermal Necrolysis: A Case Series.

Toxic epidermal necrolysis (TEN) is a dermatological process which has lacked both clear pathophysiological definition and efficacious medical treatment. This leads to metabolic dysfunction due to the inability to regulate fluid and electrolytes after the loss of skin. It is a deadly and costly disease which is associated with long lengths of stay and high-mortality rates. The depth of TEN mimics that of a partial-thickness burn. There has been documentation of successful usage of autologous skin cell suspension (ASCS) in TEN. This study expands upon our previous experience using ASCS in TEN to a series of three. Dermatology is consulted for biopsy along with the burn surgery team for wound care, where a Score for TEN is performed for risk stratification. Aggressive operative debridement is performed in the operative suite and a healthy, uninvolved donor site is harvested and processed per standard protocol. Dressings are taken down at postoperative day 4 for evaluation. The average length of stay when compared to historical data in literature is a reduction by 48%. ICU days were reduced by 64%. Cost was reduced by 54%. There was no mortality in our population of three. ASCS is both therapeutically and cost effective at treating TEN. The question of type of dressing and decision to operate is mitigated by this intervention. As an efficacious intervention, it reduces hospital stay, reduces wound cares, speeds healing, and provides a cosmetically acceptable outcome.

Inhalation Injury Grading Using Transfer Learning Based on Bronchoscopy Images and Mechanical Ventilation Period.

The abbreviated injury score (AIS) is commonly used as a grading system for inhalation injuries. While inhalation injury grades have inconsistently been shown to correlate positively with the time mechanical ventilation is needed, grading is subjective and relies heavily on the clinicians' experience and expertise. Additionally, no correlation has been shown between these patients' inhalation injury grades and outcomes. In this paper, we propose a novel inhalation injury grading method which uses deep learning algorithms in bronchoscopy images to determine the injury grade from the carbonaceous deposits, blistering, and fibrin casts in the bronchoscopy images. The proposed method adopts transfer learning and data augmentation concepts to enhance the accuracy performance to avoid overfitting. We tested our proposed model on the bronchoscopy images acquired from eighteen patients who had suffered inhalation injuries, with the degree of severity 1, 2, 3, 4, 5, or 6. As performance metrics, we consider accuracy, sensitivity, specificity, F-1 score, and precision. Experimental results show that our proposed method, with both transfer learning and data augmentation components, provides an overall 86.11% accuracy. Moreover, the experimental results also show that the performance of the proposed method outperforms the method without transfer learning or data augmentation.

Synergistic Use of Novel Technological Advances in Burn Care Significantly Reduces Hospital Length of Stay Below Predicted: A Case Series.

Length of stay is an important metric in healthcare systems, primarily because it reflects the cost of care provided. In the United States, as in many countries, inpatient hospital stays are significantly more expensive than outpatient care across all healthcare conditions,1 so earlier discharge and transition to outpatient care is crucial to help control the ever-increasing cost of healthcare. In burn patients, length of stay has traditionally been estimated at 1 day per 1% total body surface area of burn. This estimation was first described in a round table discussion in 1986.2 However, since that time there has been significant evolution in the quality of care available to burn patients, in both the operating room and ICU. The use of new harvesting techniques, synthetic dermal substitution, and autologous epidermal skin cell suspension are allowing large, deep burns to be excised and covered in much quicker time frames than historically were possible. Examples include the skin harvesting and wound debridement device for grafting and excision, biodegradable temporizing matrix as a fully synthetic dermal template, and regenerative epidermal suspension concerning cell harvesting. Although these modalities can all be used separately, we believe that using them in conjunction has allowed us to shorten the length of stay in patients with severe partial and full-thickness burns. We present an initial case series of three patients with anticipated hospital lengths of stay of 54.5, 55, and 51 days, who were ready for discharge in 37, 35, and 43 days, respectively.

Vitamin A-Induced Hypercalcemia in Burn Patients: A Case Study.

Vitamin and steroid supplementation such as oxandrolone are commonly given to speed the recovery process in severe burn injuries. Vitamin A is administered concurrently with steroids because of its pro-inflammatory and positive effects on wound healing. However, vitamin A supplementation warrants caution as hypercalcemia can result from vitamin A overdose. Our case involves an 18-year-old male injured in an oil field explosion who presented with 55% total body surface area (TBSA) partial- and full-thickness burns. Following successful resuscitation, he was given vitamin A, oxandrolone, vitamin C, and zinc sulfate as part of the standard vitamin supplementation. On hospital day (HD) 33, serum calcium levels were noted to be elevated and increased to 13 mg/dL a few days later. Parathyroid hormone and vitamin D levels were found to be within normal range, and urine analysis showed normal calcium excretion. Subsequent assessment of vitamin A levels revealed significantly elevated levels at 93 mcg/dL. Vitamin A supplementation was discontinued, and the patient was discharged on HD 42. At the 1-month follow-up, serum calcium levels were normal, which links the hypercalcemia to vitamin A overdose. This case highlights the importance of considering vitamin A overdose as a cause for asymptomatic hypercalcemia with a normal parathyroid and vitamin D workup. While routine, vitamin A supplementation in burn patients calls for assessment of both serum calcium and vitamin A levels throughout the hospital stay to prevent hypercalcemia and its negative effects.

Autologous Skin Cell Suspension Application for Toxic Epidermal Necrolysis: A Case Report.

Toxic epidermal necrolysis (TEN) is a drug-mediated disease process that mimics a partial-thickness thermal injury. It has long been treated with frequent wound dressing changes and supportive care. There has been minimal efficacious systemic therapy. The pathophysiology is poorly understood but causes necrosis of keratinocytes at the dermal-epidermal junction leading to sloughing of the epidermis. The disease is rare with high mortality rates associated with long hospital stays. This case report describes the application of autologous skin cell suspension to a patient with TEN after antihypertensive and hyperglycemic therapy. This was associated with minimal wound care and efficacious arrest of patient disease process and timely closure of wound.

Pten Regulates Cardiomyocyte Differentiation by Modulating Non-CG Methylation via Dnmt3.

The regulation of cardiomyocyte differentiation is a fundamental aspect of cardiac development and regenerative medicine. PTEN plays important roles during embryonic development. However, its role in cardiomyocyte differentiation remains unknown. In this study, a low-cost protocol for cardiomyocyte differentiation from mouse embryonic stem cells (ESCs) is presented and it is shown that Pten deletion potently suppresses cardiomyocyte differentiation. Transcriptome analysis shows that the expression of a series of cardiomyocyte marker genes is downregulated in Pten-/- cardiomyocytes. Pten ablation induces Dnmt3b expression via the AKT/FoxO3a pathway and regulates the expression of a series of imprinted genes, including Igf2. Double knockout of Dnmt3l and Dnmt3b rescues the deficiency of cardiomyocyte differentiation of Pten-/- ESCs. The DNA methylomes from wild-type and Pten-/- embryoid bodies and cardiomyocytes are analyzed by whole-genome bisulfite sequencing. Pten deletion significantly promotes the non-CG (CHG and CHH) methylation levels of genomic DNA during cardiomyocyte differentiation, and the non-CG methylation levels of cardiomyocyte genes and Igf2 are increased in Pten-/- cardiomyocytes. Igf2 or Igf1r deletion also suppresses cardiomyocyte differentiation through the MAPK/ERK signaling pathway, and IGF2 supplementation partially rescues the cardiomyocyte differentiation. Finally, Pten conditional knockout mice are generated and the role of PTEN in cardiomyocyte differentiation is verified in vivo.

Intercostal Nerve Block with Liposomal Bupivacaine vs Epidural Analgesia for the Treatment of Traumatic Rib Fracture.

BACKGROUND: Rib fractures are common among trauma patients and analgesia remains the cornerstone of treatment. Intercostal nerve blocks provide analgesia but are limited by the duration of the anesthetic. This study compares outcomes of epidural analgesia with intercostal nerve block using liposomal bupivacaine for the treatment of traumatic rib fractures. METHODS: A retrospective chart review was used to identify patients who received either epidural analgesia or intercostal nerve block with liposomal bupivacaine for the treatment of traumatic rib fractures. Patients were matched in a 1:1 ratio on age, Injury Severity Score, and number of rib fractures. Outcomes included intubations, mechanical ventilation days, ICU length of stay (LOS), hospital LOS, and mortality. RESULTS: After matching, 116 patients were included in the study. Patients receiving intercostal nerve blocks with liposomal bupivacaine were less likely to require intubation (3% vs 17%; p = 0.015), had shorter hospital LOS (mean ± SD 8 ± 6 days vs 11 ± 9 days; p = 0.020) and ICU LOS (mean ± SD 2 ± 5 days vs 5 ± 6 days; p = 0.007). There were no differences in ventilator days or mortality. Minor complications occurred in 26% of patients that received an epidural catheter for rib fractures. No complications occurred in the patients receiving intercostal nerve block. CONCLUSIONS: Patients who received intercostal nerve blocks with liposomal bupivacaine required intubation less frequently and had shorter ICU and hospital LOS compared with epidural analgesia patients. These results suggest that intercostal nerve blocks with liposomal bupivacaine might be equal or superior to epidural analgesia.

Idiopathic Autism: Cellular and Molecular Phenotypes in Pluripotent Stem Cell-Derived Neurons.

Autism spectrum disorder is a complex neurodevelopmental disorder whose pathophysiology remains elusive as a consequence of the unavailability for study of patient brain neurons; this deficit may potentially be circumvented by neural differentiation of induced pluripotent stem cells. Rare syndromes with single gene mutations and autistic symptoms have significantly advanced the molecular and cellular understanding of autism spectrum disorders; however, in aggregate, they only represent a fraction of all cases of autism. In an effort to define the cellular and molecular phenotypes in human neurons of non-syndromic autism, we generated induced pluripotent stem cells (iPSCs) from three male autism spectrum disorder patients who had no identifiable clinical syndromes, and their unaffected male siblings and subsequently differentiated these patient-specific stem cells into electrophysiologically active neurons. iPSC-derived neurons from these autistic patients displayed decreases in the frequency and kinetics of spontaneous excitatory postsynaptic currents relative to controls, as well as significant decreases in Na+ and inactivating K+ voltage-gated currents. Moreover, whole-genome microarray analysis of gene expression identified 161 unique genes that were significantly differentially expressed in autistic patient iPSC-derived neurons (>twofold, FDR < 0.05). These genes were significantly enriched for processes related to synaptic transmission, such as neuroactive ligand-receptor signaling and extracellular matrix interactions, and were enriched for genes previously associated with autism spectrum disorder. Our data demonstrate aberrant voltage-gated currents and underlying molecular changes related to synaptic function in iPSC-derived neurons from individuals with idiopathic autism as compared to unaffected siblings controls.

Modulation of microRNA expression in human lung cancer cells by the G9a histone methyltransferase inhibitor BIX01294.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of their target genes at the post-transcriptional level. In cancer cells, miRNAs, depending on the biological functions of their target genes, may have a tumor-promoting or -suppressing effect. Treatment of cancer cells with inhibitors of DNA methylation and/or histone deacetylation modulates the expression level of miRNAs, which provides evidence for epigenetic regulation of miRNA expression. The consequences of inhibition of histone methyltransferase on miRNA expression, however, have not been thoroughly investigated. The present study examined the expression pattern of miRNAs in the non-small cell lung cancer cell line, H1299 with or without treatment of BIX01294, a potent chemical inhibitor of G9a methyltransferase that catalyzes the mono-and di-methylation of the lysine 9 residue of histone H3. By coupling microarray analysis with quantitative real-time polymerase chain reaction analysis, two miRNAs were identified that showed consistent downregulation following BIX01294 treatment. The results indicate that histone H3 methylation regulates miRNA expression in lung cancer cells, which may provide additional insight for future chemical treatment of lung cancer.

Up-regulation of proliferative and migratory genes in regulatory T cells from patients with metastatic castration-resistant prostate cancer.

A higher frequency of regulatory T cells (Tregs) has been observed in peripheral blood mononuclear cells (PBMC) of patients with different types of solid tumors and hematological malignancies as compared to healthy donors. In prostate cancer patients, Tregs in PBMC have been shown to have increased suppressive function. Tumor-induced biological changes in Tregs may enable tumor cells to escape immunosurveillance. We performed genome-wide expression analyses comparing the expression levels of more than 38,500 genes in Tregs with similar suppressive activity, isolated from the peripheral blood of healthy donors and patients with metastatic castration-resistant prostate cancer (mCRPC). The differentially expressed genes in mCRPC Tregs are involved in cell cycle processes, cellular growth and proliferation, immune responses, hematological system development and function and the interleukin-2 (IL-2) and transforming growth factor-ß (TGF-ß) pathways. Studies revealed that the levels of expression of genes responsible for T-cell proliferation (C-FOS, C-JUN and DUSP1) and cellular migration (RGS1) were greater in Tregs from mCRPC patients as compared to values observed in healthy donors. Increased RGS1 expression in Tregs from mCRPC patients suggests a decrease in these Tregs' migratory ability. In addition, the higher frequency of CD4(+) CD25(high) CD127(-) Tregs in the peripheral blood of mCRPC patients may be the result of an increase in Treg proliferation capacity. Results also suggest that the alterations observed in gene expression profiles of Tregs in mCRPC patients may be part of the mechanism of tumor escape from host immune surveillance.

Biochemical characterization of a testis-predominant isoform of N-alpha acetyltransferase.

N-alpha protein acetylation, catalyzed by N-alpha acetyltransferase complex, is a common protein -modification process in eukaryotic cells. Despite its widespread occurrence, the biological significance of this modification process is still unclear. We recently discovered a novel testis-predominant isoform of the catalytic subunit of the enzyme complex. Here, we describe the biochemical characterization of this testis-predominant N-alpha acetyltransferase complex, which includes protein-protein interaction study by co-immunoprecipitation experiment and functional study by N-alpha acetyltransferase assay.

Expression of human NAA11 (ARD1B) gene is tissue-specific and is regulated by DNA methylation.

NAA10 gene encodes the catalytic subunit of N(alpha)-acetyltransferase NatA that catalyzes the acetylation of the N-termini of many eukaryotic proteins. A homologous gene called NAA11 is also present in mammalian cells. hNaa10p and hNaa11p are reported to be co-expressed in human cell cultures. In mouse tissues, however, Naa11 transcripts can only be detected in gonadal tissues whereas Naa10 transcripts are present in various tissues. We re-examined the expression of NAA11 in human cell lines and expanded the test to normal as well as cancerous human tissues. Surprisingly, we did not detect the expression of NAA11 in human cell lines that previously were reported to express it. Similar to its mouse ortholog, NAA10 displayed widespread expression in human tissues. NAA11 transcripts, however, were only detected in testicular and placental tissues. The lack of NAA11 expression was also demonstrated in eight different types of human cancerous tissues. By methylation-specific polymerase chain reaction and bisulfite sequencing, we found that the absence of NAA11 expression correlated with hypermethylation of the CpG island located at the proximal promoter of NAA11 gene. We also found that the cloned NAA11 gene promoter fragment was active when introduced into non NAA11-expressing human cells and its promoter activity was lost upon in vitro DNA methylation. Taken together, our results indicate NAA11 expression is tissue-specific and is epigenetically regulated by DNA methylation.

Genomic landscape of developing male germ cells.

Spermatogenesis is a highly orchestrated developmental process by which spermatogonia develop into mature spermatozoa. This process involves many testis- or male germ cell-specific gene products whose expressions are strictly regulated. In the past decade the advent of high-throughput gene expression analytical techniques has made functional genomic studies of this process, particularly in model animals such as mice and rats, feasible and practical. These studies have just begun to reveal the complexity of the genomic landscape of the developing male germ cells. Over 50% of the mouse and rat genome are expressed during testicular development. Among transcripts present in germ cells, 40% - 60% are uncharacterized. A number of genes, and consequently their associated biological pathways, are differentially expressed at different stages of spermatogenesis. Developing male germ cells present a rich repertoire of genetic processes. Tissue-specific as well as spermatogenesis stage-specific alternative splicing of genes exemplifies the complexity of genome expression. In addition to this layer of control, discoveries of abundant presence of antisense transcripts, expressed psuedogenes, non-coding RNAs (ncRNA) including long ncRNAs, microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), and retrogenes all point to the presence of multiple layers of expression and functional regulation in male germ cells. It is anticipated that application of systems biology approaches will further our understanding of the regulatory mechanism of spermatogenesis.