Creating memories: molecular mechanisms of CRISPR adaptation.

Prokaryotes use clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas) proteins as an adaptive immune system. CRISPR-Cas systems preserve molecular memories of infections by integrating short fragments of foreign nucleic acids as spacers into the host CRISPR array in a process termed 'adaptation'. Functional spacers ensure a robust immune response by Cas effectors, which neutralizes subsequent infection through RNA-guided interference pathways. In this review, we summarize recent discoveries that have advanced our understanding of adaptation, with a focus on how functional spacers are generated and incorporated through many widespread, but type-specific, mechanisms. Finally, we highlight future directions and outstanding questions for a more thorough understanding of CRISPR adaptation.

Cas4-Dependent Prespacer Processing Ensures High-Fidelity Programming of CRISPR Arrays.

CRISPR-Cas immune systems integrate short segments of foreign DNA as spacers into the host CRISPR locus to provide molecular memory of infection. Cas4 proteins are widespread in CRISPR-Cas systems and are thought to participate in spacer acquisition, although their exact function remains unknown. Here we show that Bacillus halodurans type I-C Cas4 is required for efficient prespacer processing prior to Cas1-Cas2-mediated integration. Cas4 interacts tightly with the Cas1 integrase, forming a heterohexameric complex containing two Cas1 dimers and two Cas4 subunits. In the presence of Cas1 and Cas2, Cas4 processes double-stranded substrates with long 3' overhangs through site-specific endonucleolytic cleavage. Cas4 recognizes PAM sequences within the prespacer and prevents integration of unprocessed prespacers, ensuring that only functional spacers will be integrated into the CRISPR array. Our results reveal the critical role of Cas4 in maintaining fidelity during CRISPR adaptation, providing a structural and mechanistic model for prespacer processing and integration.

Intermediate Cervical Plexus Block in the Management of Refractory Somatosensory Tinnitus Following Whiplash: Prospective Series in 30 Patients.

OBJECTIVE: Whiplash-associated disorder comprises of a constellation of persistent symptoms after neck trauma. Tinnitus that develops postwhiplash is termed somatosensory tinnitus. The objective is to assess the role of intermediate cervical plexus block (iCPB) in patients with somatosensory tinnitus secondary to whiplash. METHODS: Prospective service evaluation in adults with whiplash-associated disorder and concomitant somatosensory tinnitus. Patients underwent specialist otorhinolaryngology review before pain clinic referral. Patients were offered ultrasound-guided iCPB with steroids. Intensity of tinnitus was recorded on a numerical rating scale at baseline, 3 and 6 months posttreatment. Brief Pain Inventory Short Form and Hospital Anxiety Depression Scale questionnaires were also completed. RESULTS: Over a 36-month period, 32 patients with refractory somatosensory tinnitus following whiplash were offered iCPB(s). Two patients refused because of needle phobia. iCPB(s) was performed in 30 patients as an outpatient procedure. One patient (1/30, 3.3%) was lost to follow-up. Twenty-three patients (23/30, 77%) reported clinically significant reduction in intensity of tinnitus at 3 months postprocedure. Nineteen patients (19/30, 63%) reported ongoing benefit at 6-month follow-up. Six patients failed to report any benefit (6/30, 20%). CONCLUSION: The cervical plexus could play a significant role in the development of somatosensory tinnitus after whiplash. iCPB may have a role in the management of somatosensory tinnitus in this cohort.

The incidence of post cholecystectomy pain (PCP) syndrome at 12 months following laparoscopic cholecystectomy: a prospective evaluation in 200 patients.

OBJECTIVES: Post cholecystectomy pain syndrome can cause significant distress, impairs quality of life and exacerbations often result in emergency visits. Poorly controlled postoperative pain is a recognized cause of persistent postsurgical pain. Abdominal myofascial pain syndrome is an underdiagnosed cause of persistent pain in this cohort. The objective was to estimate the incidence of poorly controlled postoperative pain in the first 48 h after surgery and the likelihood of developing persistent pain at 12 months. METHODS: The patients undergoing laparoscopic cholecystectomy at a tertiary unit were consented for participation in a prospective service evaluation. A telephone review was performed at three, six and twelve months after surgery. Incidence of poorly controlled pain in the first 48 h after surgery was assessed. Patients with persistent pain were referred to the pain clinic. RESULTS: Over a six-month period, 200 patients were assessed. Eleven patients were excluded (5.5 %). Twelve patients were lost to follow-up (6.6 %, 12/189). Patient satisfaction with acute postoperative pain management was low in 40 % (76/189). Poorly controlled postoperative pain was reported by 36 % (68/189) of patients. Incidence of persistent pain was 29 % (54/189) at 12 months post-surgery. Over half of patients with persistent pain (63 %, 34/54) reported poorly controlled postoperative pain. A somatic source was diagnosed in 54 % (29/54) with post cholecystectomy pain syndrome. CONCLUSIONS: Poorly controlled postoperative pain was reported by a third of patients. Persistent pain was present in 29 % at twelve months post-surgery. Abdominal myofascial pain syndrome should be considered as a differential diagnosis in post cholecystectomy pain syndrome.

Interventional pathway in the management of refractory post cholecystectomy pain (PCP) syndrome: a 6-year prospective audit in 60 patients.

OBJECTIVES: Post cholecystectomy pain syndrome can affect over a third of patients undergoing laparoscopic cholecystectomy. Acute exacerbations can result in recurrent emergency admission with excessive healthcare utilization. Standard surgical management appears to focus on visceral aetiology. Abdominal myofascial pain syndrome is a poorly recognised somatic pathology that can cause refractory pain in this cohort. It develops as a result of trigger points in the abdominal musculature. The report describes the pathophysiology and a novel interventional pathway in the management of post cholecystectomy pain secondary to abdominal myofascial pain syndrome. METHODS: The prospective longitudinal audit was performed at a tertiary pain medicine clinic in a university teaching hospital. Over a six-year period, adult patients with refractory abdominal pain following laparoscopic cholecystectomy were included in a structured interventional management pathway. The pathway included two interventions. Intervention I was a combination of abdominal plane blocks and epigastric port site trigger injection with steroids. Patients who failed to report durable relief (>50% pain relief at 12 weeks) were offered pulsed radiofrequency treatment to the abdominal planes (Intervention II). Outcomes included patient satisfaction, change in opioid consumption and impact on emergency visits. RESULTS: Sixty patients who failed to respond to standard management were offered the pathway. Four patients refused due to needle phobia. Fifty-six patients received Intervention I. Failure rate was 14% (8/56). Forty-eight patients (48/56, 86%) reported significant benefit at 12 weeks while 38 patients reported durable relief at 24 weeks (38/56, 68%). Nine patients received Intervention II and all (100%) reported durable relief. Emergency admissions and opioid consumption were reduced. CONCLUSIONS: Abdominal myofascial pain syndrome is a poorly recognised cause of post cholecystectomy pain. The novel interventional management pathway could be an effective solution in patients who fail to benefit from standard management.

Effectiveness of Intermediate Cervical Plexus Block in Whiplash-Associated Disorder: A Prospective Observational Trial in Fifty Patients.

BACKGROUND: Whiplash trauma can result in a range of symptoms, including chronic neck pain, headache, facial pain, upper back pain, and tinnitus, which comprises whiplash-associated disorder (WAD). Intermediate cervical plexus block (iCPB) is a novel intervention that targets the upper cervical nerves and anecdotal reports suggest benefits in WAD. OBJECTIVES: We hypothesized that the cervical plexus may have a role in the pathogenesis of WAD and blocking the cervical plexus may provide analgesia. STUDY DESIGN: Prospective observational trial. SETTING: Tertiary pain medicine unit at a university teaching hospital. METHODS: Adult patients who presented with refractory chronic neck pain following whiplash were included in a prospective observational trial. The pragmatic trial studied the effectiveness of 2 sequential cervical plexus blocks (iCPB with local anesthetic [iCPB-LA] and iCPB with steroid and LA mixture [iCPB-Steroid]) in refractory chronic neck pain following whiplash. Patients who reported < 50% relief at 12 weeks after iCPB-LA were offered iCPB-Steroid. Primary outcome was "neck pain at its worst in the last 24 hours" at 12 weeks. Secondary outcomes included change in neck disability index, employment status, and mood. RESULTS: After excluding cervical zygapophyseal joint dysfunction, 50 patients underwent the iCPB-LA between June 2020 and August 2022. Five patients reported > 50% relief (durable relief) at 12 weeks and 3 patients were lost to follow-up. Forty-two patients received iCPB-Steroid. iCPB-Steroid was associated with significant reduction in neck pain, neck disability, and improvement in mood at 12 weeks when compared to the block with LA. In addition, iCPB-Steroid was associated with significant reduction in neck pain and disability at 24 weeks. Due to functional improvement, 34 patients (34/50, 78%) were able to maintain employment. LIMITATIONS: This is an open-label, observational, single-center study in a limited cohort under a single physician. Cervical facet joint dysfunction was ruled out clinically and radiologically. CONCLUSIONS: Cervical plexus may play a central role in the pathogenesis of WAD. iCPB could potentially be a treatment option in this cohort.

Intermediate Cervical Plexus Block for Refractory Bilateral Facial Pain From a Whiplash Injury in 3 Patients: A Case Report.

Bilateral facial pain is associated with temporomandibular joint dysfunction and rarely, trigeminal neuralgia. In the absence of clinical and radiological signs, a diagnosis of persistent idiopathic facial pain is often made. Standard management of persistent idiopathic facial pain includes pharmacotherapy and psychotherapy with variable therapeutic efficacy. Whiplash can result in persistent facial pain although its clinical presentation and management are poorly defined. This report includes 3 patients with refractory bilateral facial pain. A detailed review of history revealed whiplash before the onset of the symptoms. The authors present a novel intervention, an intermediate cervical plexus block that produced durable analgesia.

TNF-α differentially modulates subunit levels of respiratory electron transport complexes of ER/PR +ve/-ve breast cancer cells to regulate mitochondrial complex activity and tumorigenic potential.

BACKGROUND: Tumor necrosis factor-α (TNF-α) is an immunostimulatory cytokine that is consistently high in the breast tumor microenvironment (TME); however, its differential role in mitochondrial functions and cell survival in ER/PR +ve and ER/PR -ve breast cancer cells is not well understood. METHODS: In the current study, we investigated TNF-α modulated mitochondrial proteome using high-resolution mass spectrometry and identified the differentially expressed proteins in two different breast cancer cell lines, ER/PR positive cell line; luminal, MCF-7 and ER/PR negative cell line; basal-like, MDA-MB-231 and explored its implication in regulating the tumorigenic potential of breast cancer cells. We also compared the activity of mitochondrial complexes, ATP, and ROS levels between MCF-7 and MDA-MB-231 in the presence of TNF-α. We used Tumor Immune Estimation Resource (TIMER) webserver to analyze the correlation between TNF-α and mitochondrial proteins in basal and luminal breast cancer patients. Kaplan-Meier method was used to analyze the correlation between mitochondrial protein expression and survival of breast cancer patients. RESULTS: The proteome analysis revealed that TNF-α differentially altered the level of critical proteins of mitochondrial respiratory chain complexes both in MCF-7 and MDA-MB-231, which correlated with differential assembly and activity of mitochondrial ETC complexes. The inhibition of the glycolytic pathway in the presence of TNF-α showed that glycolysis is indispensable for the proliferation and clonogenic ability of MDA-MB-231 cells (ER/PR -ve) as compared to MCF-7 cells (ER/PR +ve). The TIMER database showed a negative correlation between the expressions of TNF-α and key regulators of mitochondrial OXPHOS complexes in basal breast vs lobular carcinoma. Conversely, patient survival analysis showed an improved relapse-free survival with increased expression of identified proteins of ETC complexes and survival of the breast cancer patients. CONCLUSION: The evidence presented in our study convincingly demonstrates that TNF-α regulates the survival and proliferation of aggressive tumor cells by modulating the levels of critical assembly factors and subunits involved in mitochondrial respiratory chain supercomplexes organization and function. This favors the rewiring of mitochondrial metabolism towards anaplerosis to support the survival and proliferation of breast cancer cells. Collectively, the results strongly suggest that TNF-α differentially regulates metabolic adaptation in ER/PR +ve (MCF-7) and ER/PR -ve (MDA-MB-231) cells by modulating the mitochondrial supercomplex assembly and activity.

The Cas4-Cas1-Cas2 complex mediates precise prespacer processing during CRISPR adaptation.

CRISPR adaptation immunizes bacteria and archaea against viruses. During adaptation, the Cas1-Cas2 complex integrates fragments of invader DNA as spacers in the CRISPR array. Recently, an additional protein Cas4 has been implicated in selection and processing of prespacer substrates for Cas1-Cas2, although this mechanism remains unclear. We show that Cas4 interacts directly with Cas1-Cas2 forming a Cas4-Cas1-Cas2 complex that captures and processes prespacers prior to integration. Structural analysis of the Cas4-Cas1-Cas2 complex reveals two copies of Cas4 that closely interact with the two integrase active sites of Cas1, suggesting a mechanism for substrate handoff following processing. We also find that the Cas4-Cas1-Cas2 complex processes single-stranded DNA provided in cis or in trans with a double-stranded DNA duplex. Cas4 cleaves precisely upstream of PAM sequences, ensuring the acquisition of functional spacers. Our results explain how Cas4 cleavage coordinates with Cas1-Cas2 integration and defines the exact cleavage sites and specificity of Cas4.